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Biochimica et Biophysica Acta 1783 (2008) 651 672

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Review
Ca 2+ -permeable channels in the hepatocyte plasma membrane
and their roles in hepatocyte physiology
Gregory J. Barritt a,, Jinglong Chen b , Grigori Y. Rychkov c
a
Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University,
G.P.O. Box 2100, Adelaide South Australia 5001; Australia
b
Cytokine Molecular Biology and Signalling Group, Division of Molecular Biology, The John Curtin School of Medical Research,
Australian National University, Canberra ACT 0200, Australia
c
School of Molecular and Biomedical Science, University of Adelaide, Adelaide South Australia 5005, Australia
Received 24 September 2007; received in revised form 16 January 2008; accepted 17 January 2008
Available online 7 February 2008

Abstract

Hepatocytes are highly differentiated and spatially polarised cells which conduct a wide range of functions, including intermediary metabolism,
protein synthesis and secretion, and the synthesis, transport and secretion of bile acids. Changes in the concentrations of Ca2+ in the cytoplasmic
space, endoplasmic reticulum (ER), mitochondria, and other intracellular organelles make an essential contribution to the regulation of these
hepatocyte functions. While not yet fully understood, the spatial and temporal parameters of the cytoplasmic Ca2+ signals and the entry of Ca2+
through Ca2+-permeable channels in the plasma membrane are critical to the regulation by Ca2+ of hepatocyte function. Ca2+ entry across the
hepatocyte plasma membrane has been studied in hepatocytes in situ, in isolated hepatocytes and in liver cell lines. The types of Ca2+-permeable
channels identified are store-operated, ligand-gated, receptor-activated and stretch-activated channels, and these may vary depending on the
animal species studied. Rat liver cell store-operated Ca2+ channels (SOCs) have a high selectivity for Ca2+ and characteristics similar to those of
the Ca2+ release activated Ca2+ channels in lymphocytes and mast cells. Liver cell SOCs are activated by a decrease in Ca2+ in a sub-region of the
ER enriched in type1 IP3 receptors. Activation requires stromal interaction molecule type 1 (STIM1), and Gi2, F-actin and PLC1 as facilitatory
proteins. P2x purinergic channels are the only ligand-gated Ca2+-permeable channels in the liver cell membrane identified so far. Several types of
receptor-activated Ca2+ channels have been identified, and some partially characterised. It is likely that TRP (transient receptor potential)
polypeptides, which can form Ca2+- and Na+-permeable channels, comprise many hepatocyte receptor-activated Ca2+-permeable channels. A
number of TRP proteins have been detected in hepatocytes and in liver cell lines. Further experiments are required to characterise the receptor-
activated Ca2+ permeable channels more fully, and to determine the molecular nature, mechanisms of activation, and precise physiological
functions of each of the different hepatocyte plasma membrane Ca2+ permeable channels.
2008 Elsevier B.V. All rights reserved.

Keywords: Hepatocyte; Liver; Ca2+ channel; Plasma membrane; TRP channel; Hormone

Abbreviations: ER, endoplasmic reticulum; SERCA, endoplasmic reticulum
(Ca2+ + Mg2+) ATP-ase; PM, plasma membrane; IP3R, inositol 1,4,5-trispho-
sphate receptor; SOC, store-operated Ca2+ channel; ISOC, store-operated channel
current; CRAC, Ca2+ release activated Ca2+ channel; STIM1, stromal interaction
molecule type 1; TRP, transient receptor potential; [Ca2+]cyt, cytoplasmic free
Ca2+ concentration; [Ca2+]er, free Ca2+ concentration in the ER; [Ca2+]mt, free
Ca2+ concentration in the mitochondria; Ca2+ext, extracellular Ca2+; PLC,
phospholipase C; VOCC, voltage-operated Ca2+ channel; DBHQ, 2,5-di-(tert-
butyl)1,4-benzohydro-quinone
Corresponding author. Tel.: +61 8 8204 4260; fax: +61 8 8374 0139.
E-mail address: Greg.Barritt@flinders.edu.au (G.J. Barritt).
0167-4889/$ - see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2008.01.016
1. Introduction
It is now about 30 years since the first clear evidence was
obtained that Ca2+ acts as an intracellular messenger in hepa-
tocytes (reviewed in [1]). Since that time the major components
of the hepatocyte Ca2+ signalling pathways have been elu-
cidated. These include the roles of endoplasmic reticulum (ER)
and mitochondria as intracellular Ca2+ stores, IP3-induced re-
lease of Ca2+ from the ER, plasma membrane Ca2+ entry and
(Ca2+ + Mg2+)ATP-ases in the ER and plasma membrane (re-
viewed in [2,3]). The nature of waves or oscillations of
652 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672
increased cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and
many of the target enzymes which are regulated by changes in
[Ca2+]cyt have been determined (reviewed in [2,3]). Changes in
[Ca2+] in hepatocytes constitute an essential intracellular sig-
nalling network which functions in unison with numerous other
signalling pathways, including the extensive protein kinase
network, in regulating hepatocyte and liver function in normal
and pathological conditions (reviewed in [37].
Recent studies of hepatocyte Ca2+ signalling have chiefly
been directed towards identifying the different pathways of Ca2+
entry across the hepatocyte plasma membrane; gaining an un-
derstanding of the molecular constituents of each entry pathway;
understanding the activation of Ca2+ entry by hormones, growth
factors and other signalling molecules; and elucidating the im-
mediate and down-steam physiological functions of each Ca2+
entry pathway (reviewed in [8,9]). These investigations have
recently been aided by the discoveries of TRP (transient receptor
potential channels) in animal cells and elucidation of their pro-
perties and likely functions [10]. The recent discovery of the
Orai1 (CRACM1) and stromal interaction molecule type 1
(STIM1) as the channel pore and Ca2+ sensor, respectively, of
Ca2+ release activated Ca2+ (CRAC) channels and some other
store-operated Ca2+ channels (SOCs) (reviewed in [11,12]) have
also contributed greatly to understanding the process of Ca2+
entry to animal cells. While it is likely that all these proteins do
play important roles in hepatocyte Ca2+ homeostasis, much is
still to be learned about the molecular nature and mechanisms
of activation of Ca2+ entry pathways in the hepatocyte plasma
membrane.
The aim of this review is to summarise current knowledge of
the nature, molecular structures and mechanisms of activation
of Ca2+-permeable channels in the plasma membranes of hepa-
tocytes, and to present knowledge of their roles in intracellular
Ca2+ homeostasis and in hepatocyte biology. Before examining
these topics, it is useful, to briefly describe the morphology and
function of hepatocytes, the main elements responsible for intra-
cellular Ca2+ homeostasis, and the mechanisms involved in the
regulation of hepatocyte membrane potential and cell volume.
2. Liver architecture and the morphology and functions of
hepatocytes
The liver plays a central role in metabolism, detoxification,
and in regulating the overall homeostasis of the body. Functions
of the liver include the metabolism of carbohydrates, fats, pro-
teins, and xenobiotic compounds, the synthesis and transcellular
movement of bile acids and bile fluid, and the synthesis and
secretion of proteins [13,14]. Within each liver lobe, the tissue
(liver parenchyma) is arranged into hexagonal-shaped lobules
each comprised of a central vein surrounded by six portal triads
[15,16]) (Fig. 1A). Each portal triad is comprised of a hepatic
vein, hepatic artery and bile duct (Fig. 1A). The predominant cell
type in the liver is the hepatocyte (parenchymal cell) which
comprises about 70% of all cells in the liver (equivalent to 90% of
the liver volume) [13,15,16]. Endothelial cells, biliary epithelial
cells (cholangiocytes), hepatic stellate cells, Kupffer cells (mac-
rophages) and oval cells also play important roles in the liver.
Hepatocytes, which are often considered as specialised epi-
thelial cells, are organised in single-cell plates (Fig. 1B). The
architecture of the liver is determined by the extracellular matrix
or scaffold. This is composed of fibrillar collagen in the portal
and central veins, and a basement membrane composed of
collagen, laminin and entactinnidogen [1517]. The hepatocyte
plates are perfused by blood from the gut (hepatic portal vein)
and heart (hepatic artery) which drains into the central vein
(Fig. 1B). The space through which blood perfuses, the sinu-
soids, are lined with sinusoidal endothelial cells (Fig. 1A). Bile
canaliculi, which are surrounded by the adjacent membranes of
two hepatocytes (Figs. 1B and 2A), collect bile fluid from each
hepatocyte and move this to the bile duct which is lined with
biliary epithelial cells (cholangiocytes) (Fig. 1A, B).
Consistent with the complex function and architecture of the
liver, hepatocytes are highly differentiated cells which exhibit
spatial polarisation and a characteristic intracellular organiza-
tion [13,1821]. Some features of the spatial polarity of hepa-
tocytes are shown in the electron micrographs in Fig. 2A and
B and schematically in Fig. 2C. Three distinct structural and
functional regions of the hepatocyte plasma membrane can be
defined: the basal or sinusoidal membrane, which faces the
blood in the sinusoids, the lateral membrane, which lines the
intercellular space, and the canalicular membrane, which faces
the canalicular lumen. Tight junctions define the canalicular
membrane region. Gap junctions permit the movement of mol-
ecules between adjacent hepatocytes [13,1821]. Much of the
hepatocyte cytoplasmic space is occupied by the ER, mitochon-
dria, other intracellular organelles (Fig. 2A). In the fed state
glycogen granules are clearly evident. The trafficking of pro-
teins and vesicles through the hepatocyte cytoplasmic space is
critical for the maintenance of hepatocyte spatial polarity and
for many hepatocyte functions, including the secretion of bile
acids and protein [18,20].
3. Ca2+ homeostasis and the role of Ca2+ as an intracellular
messenger in hepatocytes
3.1. Biochemical processes regulated by intracellular Ca2+
Changes in hepatocyte [Ca2+]cyt regulate glucose, fatty acid,
amino acid and xenobiotic metabolism, bile acid secretion,
protein synthesis and secretion, the movement of lysosomes and
other vesicles, the cell cycle and cell proliferation, and apoptosis
and necrosis [7,13,14,2224]. Changes in the concentration of
Ca2+ in intracellular organelles also play important regulatory
roles. Thus the concentration of Ca2+ in the mitochondrial
matrix ([Ca2+]mt) regulates the citric acid cycle and ATP syn-
thesis [25] and apoptosis [7], the concentration of Ca2+ in the
ER ([Ca2+]er) regulates protein synthesis and the metabolism of
xenobiotic compounds [26], and the concentration of Ca2+ in
the nucleus regulates cell proliferation [5].
An important hepatocyte-specific function regulated by intra-
cellular Ca2+ is the uptake of bile acids from the blood, excretion
of bile fluid into the bile canaliculus and the movement of bile
along the canaliculus (Fig. 2C) [13]. Hormones and bile acids
enhance the movement of bile fluid along the bile canaliculus by
 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672 653

Fig. 1. Features of liver anatomy and the organisation of hepatocytes in liver lobules. (A) A drawing of the major lobes of rat liver (a), the relationship between the
central vein and portal triads (b), and the arrangement of the hepatic vein, hepatic artery, bile duct, hepatocyte plate and sinusoidal space (c). (B) A schematic drawing
of the hepatocyte plates showing the direction of blood flow from the hepatic portal vein and hepatic artery to the central vein, and the direction of bile flow through the
bile canaliculus. (A) (b and c) re-drawn, with permission, from [15], and (B) re-drawn with permission from [206].

increasing [Ca2+]cyt, activating Ca2+-dependent myosin light
chain kinase, inducing the polymerisation of F-actin and con-
traction of the bile canaliculus [2729] (Fig. 2D).
The main extracellular signals which employ Ca2+ as an
intracellular messenger in hepatocytes are hormones, including
epinephrine, norepinephrine, vasopressin, angiotensin II, glu-
cagon, insulin, local hormones, including ATP, ADP, nitric
oxide (NO), prostaglandins, serotonin, cytokines, and extra-
cellular Ca2+ [3033]. Changes in [Ca2+]cyt are also initiated by
cell injury often mediated by the formation of reactive oxygen
species [1,22,34].
reasonably evenly throughout all regions of the ER [4,28] with
some found in ER closely associated with the plasma membrane
[3840].
Ryanodine receptors also mediate Ca2+ release from the
hepatocyte ER, although not all results are consistent with this
conclusion [37,4143]. The results of recent experiments, which
employed RT-PCR to identify ryanodine receptor mRNA, have
shown that hepatocytes possess a truncated form of the type 1
ryanodine receptor, and have provided evidence that this re-
ceptor plays a role in amplifying IP3-induced Ca2+ release from
the ER [44].

3.2. Molecular components of hepatocyte Ca2+ homeostasis
The proteins chiefly responsible for the regulation of [Ca2+]cyt,
[Ca2+]er and [Ca2+]mt in hepatocytes are summarised in Fig. 3.
These are the main components of the hepatocyte Ca2+ signalling
tool kit [35]. Rat hepatocytes express type 1 (20%) and type 2
(80%) inositol 1,4,5 trisphosphate receptors (IP3Rs) with pos-
sibly a small amount (b1%) of type 3 IP3Rs [28,36,37]. In
hepatocytes, type 2 IP3Rs are expressed chiefly in the peri-
canalicular region and are responsible for the initiation of waves
of increased [Ca2+]cyt originating from this region [4,28]. As
discussed in more detail below, type 1 IP3R are distributed
3.3. Hepatocyte Ca2+ signals are often encoded by the frequency
of Ca2+ waves (oscillations)
Physiological concentrations of epinephrine, vasopressin,
ATP, prostaglandins and some other hormones induce repetitive
increases in [Ca2+]cyt in isolated hepatocytes [4549]. Hor-
mone-induced increases in [Ca2+]cyt in single hepatocytes are
observed as waves of increased [Ca2+]cyt, with each wave
beginning at a fixed point in the cell [4547]. In hepatocyte
couplets waves of [Ca2+]cyt originate at the bile canalicular
region [28]. Increasing the hormone concentration increases the
frequency of the oscillations [45,48,50,51]. The strength of the
654 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672

Fig. 2. Some features of the morphology and spatial polarity hepatocytes. (A) Transmission electron micrograph of rat hepatocytes in situ, showing organisation of the
cytoplasmic space and organelles around the bile canaliculus. The scale bar represents 2 m (M. Teo, M. van Baal, M. Scheisser, A. Wittert, R. Padbury and G. Barritt,
unpublished results). The abbreviations are: LI, lipid inclusions; lateral PM, lateral plasma membrane; JC, junctional complex; BC, bile canaliculus; P, peroxysome; M,
mitochondria; and S, sinusoid. (B) Transmission electron micrograph of an isolated rat hepatocyte couplet showing a bile canaliculus located between the two cells.
From [207] with permission. (C) A scheme showing the pathways of bile acid movement and vesicle trafficking in hepatocytes within the hepatocyte plate. (D) A
scheme showing the role of [Ca2+]cyt in regulating F-actin movement and contraction of the bile canaliculus.

hormone-initiated Ca2+ signal is thought to be determined by
the frequency of [Ca2+]cyt oscillations [25,52]. Hormone-
induced frequency-modulated waves of increased [Ca2+]cyt are
also observed in hepatocytes in situ in perfused livers [46,
47,50]. In liver lobules, the strength of hormonal signals which
employ oscillations in [Ca2+]cyt is thought to be conveyed to
hepatocytes located in different regions of the lobule by the rate
of propagation of the wave of increased [Ca2+]cyt (reviewed
in [3]).
When isolated hepatocytes are incubated in the absence of
extracellular Ca2+ (Ca2+ext) hormone-induced oscillations in
[Ca2+]cyt decline to zero after 510 minutes [49]. This is likely
due to the transport of a small amount of Ca2+ out of the cell
during each oscillation (cf [53]), as shown also for pancreatic
acinar cells [54], resulting in a slow depletion of Ca2+ in the ER
to a level where there is insufficient to allow [Ca2+]cyt oscilla-
tions. During hormone-induced [Ca2+]cyt oscillations an in-
crease in Ca2+ entry is required in order to maintain sufficient
Ca2+ in the ER. This is likely to be mediated by store-operated
Ca2+ channels (SOCs) [55] and possibly by some other types
of Ca2+ entry channels.
4. Cytoplasmic Ca2+ and the regulation of hepatocyte
membrane potential and cell volume
Membrane potential is the main driving force for Ca2+ entry
through Ca2+-permeable channels in the plasma membrane
[10,56,57]. Furthermore, in hepatocytes changes in [Ca2+]cyt are
involved in the regulation of both membrane potential and cell
volume [5860]. Therefore, in a discussion of the nature and
function of plasma membrane Ca2+ channels in hepatocytes, it
is relevant to consider the main factors which maintain and
regulate membrane potential and cell volume.
In response to increases in the concentrations of metabolites
and bile acids in the portal blood following the digestion and
absorption of food, hepatocytes take up considerable quantities of
glucose, amino acids, bile acids and other organic molecules, as
well as Na+ and other inorganic ions from the blood. This is
 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672 655

cells, membrane potential in hepatocytes is set to a negative

value by the K+ conductance [68]. The K+ equilibrium potential
is set by the activity of the (Na+ + K+)-ATP-ase which maintains
the intracellular Na+ and K+ concentrations at low and high
values, respectively, relative to those in the blood (Fig. 4).
However, in hepatocytes there is a significant deviation of the
observed membrane potential from the predicted K+ equili-
brium potential. This suggests that Cl and Na+ conductances
also contribute to determining the observed membrane potential
[59,61] (Fig. 4).
Ca2+ plays an important role in regulating hepatocyte mem-
brane potential through a variety of Ca2+-dependent pathways
that control the activity of different types of plasma membrane
ion channels and transporters [58,59,69]. In general, the mole-
cular identities of the channels that maintain the hepatocyte
membrane potential are largely unknown, and the regulation of
hepatocyte membrane conductance by Ca2+ is not well under-
stood. Several types of Ca2+-regulated channels have been
reported in hepatocytes in a variety of species. These are Ca2+-
activated small conductance K+ channels (SK), Ca2+-dependent
non-selective cation channels which, under physiological con-
ditions, mainly provide a pathway for Na+ entry, and Ca2+-
dependent Cl channels [58,59,62,69]. Some of these channels
are expressed at different levels in different species. For
Fig. 3. The major elements which regulate the distribution and movement of
intracellular Ca2+ in hepatocytes. The plasma membrane Ca2+ entry pathways are
Ca2+-selective store-operated Ca2+ channels (SOCs), receptor-activated Ca2+-
permeable channels (listed in Tables 2 and 3) and ligand-gated Ca2+-permeable
channels. Ca2+ outflow across the plasma membrane is mediated by the plasma
membrane (Ca2+ + Mg2+)ATP-ases, PMCA1 and PMCA2w [208], with a
possible contribution from PMCA4b [209], and by the Na+-Ca2+ exchanger
[2,210,211]. Ca2+ uptake by the ER is mediated by the ER (Ca2+ + Mg2+)ATP-ase
(SERCAs) and Ca2+ outflow from the ER by types 1 and 2 IP3R, [28,36] and
ryanodine receptors [44]. Ca2+ uptake by mitochondria is mediated by an
electrogenic Ca2+ uniporter and Ca2+ outflow by Na+/Ca2+ and H+/Ca2+ anti-
porters (reviewed in [2,3,8]. Golgi also possess IP3R and (Ca2+ + Mg2+)ATP-ases
[212]. Numerous Ca2+ binding proteins are present in the cytoplasmic space and
in organelles.

associated with the inflow of water to hepatocytes. Bile acids,

Na+, and other components of bile fluid are transported across
hepatocytes and excreted into the bile canaliculus. These pro-
cesses create considerable osmotic forces, which, if not countered,
would greatly alter the hepatocyte volume. The maintenance of a
constant cell volume under these conditions requires mechanisms
which tightly control the movement of ions across the sinusoidal,
basolateral and canalicular domains of the hepatocyte plasma
membrane. The process is termed regulated volume decrease
[59]. When hepatocytes are subjected to osmotically-induced cell
shrinkage, the recovery process is termed regulatory volume
increase. The major pathways involved in regulating volume are
K+ and Cl channels, K+, Na+, and Cl co-transporters and the
(Na+ + K+)ATP-ase [59] (Fig. 4).
Measurements of the resting membrane potential of hepa- Fig. 4. The major plasma membrane channels, transporters and co-transporters
tocytes in situ in the perfused liver using sharp electrodes have involved in the maintenance of the membrane potential, the electrochemical
given values between 30 and 40 mV [6065]. In isolated gradient across the plasma membrane, and cell volume. The activity of several
channels is regulated by [Ca2+]cyt, and [Ca2+]cyt plays an important role in
hepatocytes, reported values range between 20 mV and 50 to regulating cell volume. Since the plasma membrane potential contributes to the
70 mV [9,60,66,67], reflecting, perhaps, differences in expe- driving force for Ca2+ entry through SOCs, changes in membrane potential will
rimental conditions. As in the case of the majority of animal affect the amount of Ca2+ which enters the cell through open SOCs.
656 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672
example, Ca2+-activated K+ channels are expressed in rabbit
and guinea pig hepatocytes but not in rat hepatocytes [60].
Depending on which channels are activated and the relative
sizes of the corresponding conductances, different effects of
changes in [Ca2+]cyt on membrane potential can be expected.
Under physiological conditions, changes in hepatocyte
[Ca2+]cyt, membrane potential, and cell volume occur in re-
sponse to the uptake of metabolites and bile acids and to the
binding of hormones and other extracellular signals to plasma
membrane G-protein- or tyrosine kinase- coupled receptors
[59,6973]. Often, Ca2+-mobilising hormones such as ATP,
epinephrine and vasopressin, cause characteristically different,
or even opposite, effects on hepatocyte volume (reviewed in
[59]). This indicates the presence of other signalling pathways
which add to, or modulate, the effects of changes in [Ca2+]cyt.
Glucagon, a pancreatic hormone that regulates glucose re-
lease in liver, hyperpolarizes hepatocytes and induces cell
shrinkage [61,74]. The hyperpolarization was found to be inhi-
bited by blockers of K+ channels and by ouabain, suggesting an
involvement of K+ channels and the (Na+ + K+)-ATP-ase in
glucagon action [59]. Glucagon-induced hyperpolarisation was
also found to be blocked by the phospholipase C (PLC)
inhibitor, U73122, and by Gd3+, an inhibitor of some plasma
membrane Ca2+-permeable channels, suggesting requirements
for PLC and an increase in [Ca2+]cyt [75]. Activation by glucagon
of Ca2+ entry through SOCs [9], in addition to the glucagon-
induced increase in cAMP, is the most likely cause of the ob-
served glucagon-induced activation of Ca2+-dependent K+
conductance and subsequent hyperpolarization.
The activation by glucagon of the K+ conductance alone,
however, is unlikely to induce noticeable changes in cell volume
in the absence of the activation of a significant movement of ions
of another kind. Since hepatocytes have low resting Na+, K+ and
Cl conductances [59,61] a small K+ efflux will bring membrane
potential close to the K+ equilibrium potential and stop any
further K+ efflux. However, in addition to activating Ca2+
channels, glucagon also activates a Cl conductance similar to
that activated by hepatocyte swelling [9]. This requires the cyclic
AMP binding protein, Epac, as well as Ca2+ acting as a co-factor
[9]. Activation of the Cl efflux by glucagon simultaneously
with activation of the K+ efflux through SK channels is the likely
cause of the glucagon-induced hepatocyte shrinkage.
Interestingly, another hormone epinephrine and its analogue
phenylephrine, which activates Ca2+ entry and increases K+ and
Cl conductances, causes hepatocyte swelling [59]. Cell swell-
ing is usually associated with Na+ entry but, if the cell is
depolarised, it may also possibly be associated with Cl entry.
The activation of non-selective cation channels by Ca2+ mo-
bilising hormones, which would provide an entry pathway for
Na+, has been shown in hepatocytes and liver cell lines [76].
Whether or not [Ca2+]cyt regulates these non-selective cation
channels under physiological conditions is not clear. Other
causes of hepatocyte swelling, which may, or may not, be
related to changes in [Ca2+]cyt, could include the activation of
one or more transporters for amino acids and/or bile acids.
The above discussion emphasises the interrelationships be-
tween hormone action, [Ca2+]cyt, membrane potential and cell
volume in hepatocytes. Often, in experiments designed to in-
vestigate Ca2+ entry, the membrane potential is not controlled
and might change substantially during the experiment. This, in
turn, may lead to changes in the observed rate of Ca2+ entry. For
voltage-independent Ca2+ channels, membrane hyperpolarisa-
tion causes an increase in Ca2+ entry due to an increased driving
force. In contrast, for voltage-dependent Ca2+ channels, mem-
brane hyperpolarisation may cause a decrease in Ca2+ entry,
despite a larger driving force. The decrease is due to a reduction
in open probability of the channels. For the same reasons,
membrane depolarisation, induced, for example, by Na+ entry
through activated TRP channels [77], may also have drastic
effects on Ca2+ entry [57]. Thus, in studies of Ca2+ entry to liver
cells using techniques other than patch clamp recording, it is
important to evaluate and take account of possible changes in
membrane potential.
5. Overview of hepatocyte plasma membrane
Ca2+ -permeable channels
Ligand-gated, store-operated (SOC), receptor-activated, and
stretch-activated Ca2+-permeable channels are expressed in
hepatocytes and in liver cell lines. No voltage-operated Ca2+
channels (VOCCs) have been detected [59,66,78,79]. Ligand-
gated Ca2+-permeable channels are defined as plasma mem-
brane channels activated by the binding of an extracellular
signal molecule (hormone, neurotransmitter, or growth factor)
to the extracellular domain of the channel protein or channel
subunit complex. SOCs are defined as plasma membrane Ca2+
channels activated by a decrease in the concentration of Ca2+
in the ER. Under physiological conditions this occurs when
G-protein- or tyrosine kinase-coupled receptors are activated
by the binding of a hormone or other agonist leading to the
activation of PLC or PLC, the formation of IP3, and IP3-
and Ca2+ -induced release of Ca2+ from the ER. SOCs can be
activated experimentally using inhibitors of the endoplasmic
reticulum (Ca2+ + Mg2+) ATP-ase (SERCA) such as thapsi-
gargin or 2,5-di-(tert-butyl)-1,4-benzohydro-quinone (DBHQ)
which induce the release of Ca2+ from the ER.
Receptor-activated Ca2+-permeable channels, as defined here,
are a heterogeneous group of channels activated by the binding of
a hormone or other agonist to a G-protein or tyrosine kinase-
coupled receptor. The receptor protein is separate from the
channel protein and in most cases activation involves, or is likely
to involve, the generation of an intracellular messenger which
binds to a site on the cytoplasmic domain of the channel protein,
leading to activation of the channel. Activation may also involve
proteinprotein interaction. The activation of receptor-activated
Ca2+-permeable channels would not be expected to depend on a
decrease in [Ca2+]er. Stretch-activated (mechano-sensitive) Ca2+-
permeable channels are defined here as those channels in which
there is a direct transduction of mechanical force into channel
opening.
Liver cell plasma membrane Ca2+-permeable channels have
been studied in the perfused liver, isolated hepatocytes, and in
liver cell lines (Table 1). Most liver cell lines are derived from
hepatocyte precursors and represent hepatocytes with different
 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672
Table 1
Hepatocyte preparations and liver cell lines available for the study plasma membrane Ca2+-permeable channels
Hepatocyte preparation or liver cell line References
(examples only)
Perfused liver [96]
Freshly isolated hepatocytes in suspension [1,185]
Isolated hepatocytes maintained in primary [82,99,166]
culture for 272 h
Freshly isolated hepatocyte couplets, [29]
triplets and clusters
H4-IIE rat liver cells (derived from [97,98]
Reuber hepatoma)
HTC rat liver cells (derived from rat hepatoma) [150]
HepG2 human liver cells (derived from a [152]
liver carcinoma)
Clone 9 cells [34]
WIF B cells (derived by the fusion of a rat [80] [81]
hepatoma cell line with a human fibroblast
cell line
degrees of de-differentiation. While liver cell lines offer a
number of technical advantages for studies of the molecular
nature of Ca2+ permeable channels, they do not exhibit all the
properties of primary hepatocytes, including all the features of
the spatial polarity of hepatocytes in situ, or the spatial polarity
of freshly-isolated hepatocyte couplets [19]. Moreover, for
many signalling proteins, including hormone receptors, Ca2+
channels, and IP3Rs, the relative amount of a given protein
expressed in a liver cell line may differ considerably from that
expressed in isolated hepatocytes [19,80,81]. To fully under-
stand the structure, mechanisms of activation, and physiological
functions of hepatocyte plasma membrane Ca2+-permeable
channels, it is desirable to ultimately study the properties of
these channels in cells which most closely reflect the intra-
cellular organisation of hepatocytes in the intact liver.
6. P2X ATP-activated ligand-gated Ca2+-permeable
channels
A liver cell P2X purinergic Ca2+-permeable channel activated
by ATP has been identified and partially characterised using
patch clamp recording and the intracellular fluorescent Ca2+
sensor fluo-3 in freshly-isolated guinea pig hepatocytes [82].
These channels may contribute to the mechanisms by which
ATP, acting as a local hormone, alters hepatocyte functions
following ischaemia reperfusion injury or in response to other
toxic insults to the liver [7].
7. Store-operated Ca2+ channels
7.1. Store-operated Ca2+ channels in hepatocytes and liver cells
Many studies have shown that a SERCA inhibitor or IP3
(introduced by micro injection or generated by addition of a
657
Comments
Permits the study Ca2+ entry processes in hepatocytes in situ in a fully spatially
polarised state, and the measurement of intercellular Ca2+ signals, but technically
more difficult than experiments with isolated hepatocytes.
Hepatocytes are fully differentiated but have lost some spatial polarity upon
removal from the liver
Provide a reasonable representation of hepatocytes in situ, but undergo some
modification of protein expression and de-differentiation
Retain greater spatial polarity. Permit studies of intercellular Ca2+ signals and
roles of gap junctions.
Offer technical advantages for measurement of Ca2+ entry and cell transfection,
but have the disadvantage that they are partly de-differentiated or altered hepatocytes.
WIF B cells exhibit many features of hepatocytes including retention of spatial polarity,
canalicular structures and secretion of bile acids. They express all three subtypes of IP3R
(cf hepatocytes which expresses types 1 and 2 IP3R) but these appear not to be
homogenously distributed. May present difficulties with respect to loading cytoplasmic
space with fura-2.
hormone) will initiate the activation of Ca2+ entry to hepa-
tocytes and liver cell lines [8394]. Ca2+ entry was assessed by
measuring increases in [Ca2+]cyt using an intracellular fluor-
escent Ca2+ sensor such as fura-2, 45Ca2+, or, in the case of
perfused liver, a Ca2+ electrode to measure extracellular [Ca2+].
The results of patch clamp recording also provided evidence for
SERCA inhibitor- and IP3-initiated Ca2+ entry [95]. Since
SOCs have often been functionally defined as channels which
are activated by treatment of cells with SERCA inhibitor or IP3
[57] Ca2+ entry in response to these agents has been attributed to
SOCs.
From the results of some earlier studies with IP3 and SERCA
inhibitors it was suggested that more than one type of SOC may
be expressed in hepatocytes and liver cell lines [86,88,91,96].
However, in the majority of these studies the nature of the Ca2+
entry pathway involved was not clearly defined. In recent patch
clamp experiments only one type of SOC, a highly Ca2+-
selective channel similar to CRAC channels, could be detected
[9799]. It is possible that in some studies IP3 and SERCA
inhibitors may have initiated the activation of non-SOCs. Thus
IP3 may activate another type (non-store-operated) of plasma
membrane Ca2+-permeable channel in hepatocytes [85]. Thap-
sigargin can, under appropriate circumstances, initiate the ac-
tivation of Ca 2+ entry through Ca 2+ -permeable channels
activated by Ca2+ or by metabolites of arachidonic acid (formed
by Ca2+-activated phospholipase A2). Moreover, TRPV1 chan-
nels can be activated by the thapsigargin-induced formation of
anandamide, a potential endogenous activator of TRPV1 [100].
It is also possible that SERCA inhibitors can activate Na+ entry
through TRP channels or other non-selective cation channels
which, in turn, leads to Ca2+ entry through the Na+-Ca2+ ex-
changer working in reverse mode [101]. Thus in some studies of
hepatocytes and liver cells which have employed a SERCA
inhibitor or IP3 to initiate plasma membrane Ca2+ entry, one or
658 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672

more non-store-operated pathways of Ca2+ entry may have been
activated in addition to SOCs.
Studies with the H4-IIE rat liver cells and rat hepatocytes
using patch clamp recording in the whole cell mode have
characterised the Ca2+-permeable channels activated by thapsi-
gargin and IP3 [9799]. The SOCs exhibit a high selectivity for
Ca2+ compared with monovalent cations and exhibit properties
similar, or identical, to those of the CRAC channels found in
lymphocytes and mast cells [9799]. This conclusion was based
on a comparison of time courses of activation, current ampli-
tudes, dependence on [Ca2+]ext, conductance for Ba2+ compared
with Ca2+, and inhibition by La3+, Gd3+ and 2-aminoethyl
diphenylborate (2-APB). Ca2+ entry, measured by whole cell
patch clamp recording, through SOCs in rat hepatocytes can be
activated by physiological concentrations of vasopressin and
ATP [99] (Fig. 5). Taken together, these results provide evidence
that (i) there is only one type of SOC in rat hepatocytes, (ii) the rat
hepatocyte SOCs are highly Ca2+-selective channels essentially
identical to CRAC channels in mast cells and lymphocytes, and
(iii) the channel detected by electrophysiological techniques can
be activated by physiological concentrations of hormones in rat
hepatocytes. As discussed above, there are considerable
differences in the proteins expressed in hepatocytes from
different species, and in different liver cell lines. The majority
of studies have been conducted with rat hepatocytes and rat liver
cell lines and it is possible, or likely, that different types of SOCs
could be expressed in hepatocytes from other species.
Liver cells also express TRPM7 channels [102,103] (Rych-
kov, G., Litjens, T., Chen, J. L. and Barritt, G., unpublished
results) and it has been pointed out that the current through
TRPM7 channels can be difficult to distinguish from that through
CRAC channels [104]. However, in patch clamp experiments
divalent cation entry through Ca2+-selective SOCs in liver cells
has been measured in the presence of intracellular Mg2+. Since
intracellular Mg2+ inhibit TRPM-7 [104], it is unlikely that Ca2+
entry through TRPM-7 channels would have interfered with
characterisation of the SOC in hepatocytes and liver cells [99].
7.2. Properties of liver cell store-operated Ca2+ channels
The permeability sequence for the movement of cations
through liver cell SOCs is Ca2+ N Ba2+ N Sr2+ N Na+ N Cs+ [97].

Fig. 5. Activation of the SOC current, ISOC, by vasopressin in freshly-isolated rat hepatocytes attached to glass coverslips. (A) Development of ISOC initiated by 20 nM
vasopressin. Open symbols are the amplitudes of the inward current of the individual cells at 118 mV. Solid lines represent fits of the Boltzmann curve to the
experimental data. (B) Averaged results of the data presented in the panel A. (C) Averaged leak-subtracted IV plot of the current activated by the application of 20 nM
vasopressin to the external solution (n = 5). (D) Leak-subtracted current traces obtained in response to 200 ms steps to 118 mV in the presence of vasopressin. Data
was averaged from seven cells. Taken from (Rychkov et al 2005), with permission.
 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672
The permeability of liver cell SOCs for Mn2+ has not been
directly tested in patch clamp experiments, although it is known
that the permeability of Mn2+ through CRAC channels is low
[105]. The results of earlier work which employed Mn2+ and
fura-2 to measure Mn2+ entry, suggest that liver cell SOCs can
admit Mn2+ [90,93] although it is possible that the Mn2+ entry
observed may have been through non-store-operated pathways.
Liver cell SOCs are partially blocked by Co2+ and Cd2+ and
completely blocked by Zn2+, Gd3+ and La3+. The most potent
blocking agents are Gd3+ and La3+ which give complete block
at about 2 M in the presence of 10 mM Ca2+ext [97,99]. Results
of earlier studies of the inhibition of Ca2+ entry (measured using
fura-2) in hepatocytes by different trivalent cations led to the
conclusion that there is some similarity between the pore of the
hepatocyte SOCs and the pore of the highly Ca2+-selective
VOCCs [106].
Ca2+ entry through liver cell SOCs is inhibited by 2-aminoethyl
diphenylborate (2-APB) (S0.5 approx. 10 M) [107], SK&F96365
[78,93], arachidonic acid [108], the PLC inhibitor U73122
[109,110], and isotetrandrine and tetrandrine [108]. In addition to
providing some pharmacological characterisation of liver cell
SOCs, these results indicate that caution should be exercised in
employing U73122, an inhibitor of PLC [111] and isotetrandrine
and tetrandrine (inhibitors of phospholipase A2 [112]) in studies
of the regulation of Ca2+ entry. In H4-IIE rat liver cells, VOCC
antagonists, including verapamil, nifedipine, nicardipine and the
dihydropyridine analogues AN406 and AN1043, inhibit thapsi-
gargin-stimulated Ca2+ entry measured using a fluorescent intra-
cellular Ca2+ sensor [78] (cf [113]. However, the concentrations
of the VOCC antagonists used were considerably higher than
those which inhibit VOCCs in excitable cells suggesting that
their action on liver cell SOCs is indirect and/or non-specific.
There is some evidence to indicate that calmodulin is in-
volved in the regulation of liver cell SOCs. In rat hepatocytes,
thapsigargin-initiated Ca2+ entry was found to be inhibited by
the calmodulin antagonists calmidazolium and CGS 9343B
when the antagonists were added before (but not after) thap-
sigargin, leading to the conclusion that calmodulin is required
for Ca2+ entry [83]. In H4-IIE rat liver cells, thapsigargin-
initiated Ca2+ entry and release from intracellular stores were
observed to be inhibited by the calmodulin antagonists W7,
W13 and calmidazolium [78]. No inhibition was observed with
another calmodulin antagonist, KN62. Substantial inhibition of
Ca2+ entry by calmidazolium was only observed when this was
added before thapsigargin [78]. It was concluded that calmo-
dulin is not involved in the activation of Ca2+ entry. While the
results of some of these experiments suggest that calmodulin
may be required for liver cell SOC activation, in both studies
Ca2+ entry was measured using a fluorescent intracellular Ca2+
sensor and the nature of the Ca2+ entry channel was not defined
by patch clamp recording.
The results of patch clamp studies with H4-IIE rat liver cells
have provided evidence that the fast inactivation of the SOC
Ca2+ current, ISOC, is a calmodulin- and Ca2+-dependent pro-
cess, similar to the Ca2+-dependent fast inactivation of CRAC
channels [98]. Over-expression of either a calmodulin inhibitor
peptide or a mutant form of calmodulin lacking functional EF
659
hand domains reduced the fast component of liver cell ISOC
inactivation. However, no effect of the calmodulin antagonists
Mas-7 and calmidazolium was detected. It was concluded that
calmodulin is responsible for at least part of the Ca2+-dependent
inactivation of liver cell ISOC. Moreover, the possibility was raised
that calmodulin is tethered to the SOC protein itself and hence is
protected from the actions of calmodulin inhibitors (cf the mecha-
nism by which calmodulin regulates L-type VOCCs [114]).
7.3. Likely roles of STIM and Orai (CRACM) proteins in liver
cell store-operated Ca2+ channels
The results of recent experiments employing a wide variety
of approaches and techniques have led to the conclusion that a
member of the Orai (CRACM) family of proteins constitutes the
pore of SOCs in mast cells, lymphocytes and in some other cell
types (reviewed in [11,12,115]). Other experiments have shown
that STIM1 located in the ER constitutes the Ca2+ sensor which
detects the decrease in [Ca2+]er and conveys this information to
Orai leading to activation of the channel and Ca2+ entry. It is
presently thought that this involves the movement of some
STIM to ER-plasma membrane junctions leading to an inter-
action between STIM, located in the ER, and Orai, located in
the plasma membrane (reviewed in [11,12,115,116]). It is not
yet completely clear whether there is a direct interaction be-
tween the STIM protein and the Orai protein, or an interaction
involving additional proteins. Further, proteins other than Orai
and STIM are likely to have roles in the activation mechanism.
Other experiments suggest that the localisation of Orai and
STIM and the Ca2+ entry channel may create domains of in-
creased [Ca2+]cyt at specific locations under the plasma mem-
brane [115].
The results of recent experiments with liver cells indicate that
it is likely that STIM is required in the mechanism of SOC
activation. Thus, it has been shown using H4-IIE rat liver cells
that the siRNA-mediated knockdown of STIM1 caused a
substantial reduction in the amplitude of ISOC initiated by IP3 or
thapsigargin [110]. Treatment of H4-IIE cells with thapsigargin
leads to a re-distribution of STIM1 to puncta, as assessed using
cells transfected with GFP-STIM1 and by imaging endogenous
STIM1 by immunofluorescence (Castro, J., Jones, L., Litjens,
T., Barritt, G. and Rychkov, G., unpublished results). The pu-
tative roles of STIM1 and Orai1 in the activation of liver cell
SOCs are shown schematically in Fig. 6A.
The proposed mechanism of activation of liver cell SOCs
involving the interaction of STIM1 with Orai1 at ER-plasma
membrane junctions requires that such junctions are normally
present in hepatocytes or are formed upon depletion of Ca2+ in
the ER. Evidence for a close association of some ER with the
plasma membrane in hepatocytes comes from previous sub-
cellular fractionation experiments which generated highly puri-
fied plasma membrane fractions and provided evidence that
specialised sub-regions of the ER are located close to the plasma
membrane [38,40]. The presence of ER close to the plasma
membrane in hepatocytes is not readily apparent from inspec-
tion of electron micrographs, although areas of the ER which
come close to the plasma membrane can be seen (Fig. 6B). ER-
660 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672

Fig. 6. Proposed molecular and spatial organization of store-operated Ca2+ channels and their activation pathways in liver cells. (A) Schematic representation of some
of the proteins and organelles thought to be involved in the activation of SOCs in hepatocytes and liver cell lines. It is proposed that activation of SOCs requires sub-
regions of the ER which are in close proximity to the plasma membrane and form ER-plasma membrane junctions. These ER-sub-regions are enriched in type1 IP3R. It
is proposed that while each ER sub-region communicates with the bulk of the ER, the movement of Ca2+ between the sub-regions and the bulk of the ER is slow. The
steps in the activation of SOCs are proposed to be: the initiating decrease in [Ca2+] in the lumen of the ER induced by IP3 (physiological) or a SERCA inhibitor
(experimental), dissociation of Ca2+ from the luminal domain of the Ca2+ sensor STIM1, a conformational change in STIM1, oligomerisation of STIM1, relocalisation
of STIM1 in the ER, interaction of STIM1 in close proximity to ER-plasma membrane junctions with CRACM1/Orai1, a conformational change and increase the
probability of opening of the Orai1 channel. Other proteins (as yet unidentified) are likely to be involved. The F-actin cytoskeleton, regulated in part by Gi2 and
PLC1 are thought to play permissive roles in the activation pathway. Ca2+ which moves through SOCs into the ER-plasma membrane junction may cause a local
increase in [Ca2+]cyt at the mouth of the channel, before being transported directly to the lumen of the ER via SERCA pumps and to mitochondria. (B) Transmission
electron micrograph showing the ER in a part of an isolated rat hepatocyte near the plasma membrane with regions of the ER in the vicinity of the plasma membrane
(Wang, Y.J., Gregory, R.B., and Barritt, G.J., unpublished results). The abbreviations are: PM, plasma membrane; and ER, endoplasmic reticulum.

plasma membrane localisations have been observed by electron
microscopy in some other cell types (reviewed in [116]).
It has been proposed that a TRP (transient receptor potential)
protein, possibly TRPC1, TRPC3, TRPC4, TRPV5 and/or
TRPV6, constitutes the pore of SOCs in some types of animal
cells (reviewed in [57,117,118]. Some of these TRP proteins are
expressed in liver cells (Table 3). Ectopic expression of hTRPC1
in H4-IIE rat liver cells or knockdown of endogenous TRPC1
proteins using siRNA did not cause large changes in thapsi-
gargin-stimulated Ca2+ entry (assessed using a fluorescent Ca2+
sensor and patch clamp recording), indicating that it is unlikely
that the TRPC1 peptide constitutes SOCs in rat liver cells
[119,120]. However, a role for TRPC1 in forming or modulating
liver cell SOCs is not excluded. For example, several recent
studies have provided evidence that TRP polypeptides interact
with STIM and/or Orai polypeptides [118,121124]. As de-
scribed above, in patch clamp recording experiments only one
type of SOC can be detected in rat liver cells and this has a high
selectivity for Ca2+ comparable to that of CRAC channels
in lymphocytes and mast cells. The Ca2+-permeable channels
formed by TRPC1 polypeptides and by most other TRP
polypeptides have a relatively low selectivity for Ca2+ compared
with Na+ [10,56]. This comparison provides further evidence
that it is unlikely that any of the known TRP polypeptides
constitutes the Ca2+-selective SOCs found in rat hepatocytes and
liver cells, although TRP polypeptides may contribute to SOCs
in hepatocytes from some species.
7.4. Roles of an endoplasmic reticulum sub-region and type 1
IP3 receptors
Several experimental approaches have addressed the ques-
tion of whether all of the ER or a sub-component of the ER is
required for the activation of SOCs in liver cells. In hepatocytes
in situ, in freshly-isolated hepatocytes, and liver cell lines, the
ER is found to extend throughout most of the cytoplasmic space
[125]. The results of several experimental approaches suggest
that a small region of the ER, rather than the whole ER, is all
that is necessary for liver cell SOC activation, and that this sub-
region of the ER is enriched in type 1 IP3R. When microinjected
into freshly-isolated hepatocytes, a monoclonal anti-type 1 IP3R
antibody, which in other studies was shown to inhibit Ca2+
release mediated by type 1 IP3R, was found to inhibit hormone-
and thapsigargin-induced Ca2+ entry with little effect on the
release of Ca2+ from intracellular stores [126]. The microinjec-
tion of a relatively low concentration of adenophostin A, which
has a high affinity for IP3Rs relative to that of IP3, induced near-
maximal activation of Ca2+ entry with little detectable release of
Ca2+ from intracellular stores [126]. The results of experiments
in which IP3 analogues selective for either type 1 or type 2 IP3R
were microinjected to rat hepatocytes indicate that type 1 IP3R
are preferentially involved in SOC activation [127].
As mentioned above, the results of immunofluorescence
experiments conducted with spatially polarised rat hepatocytes in
situ, and with isolated hepatocyte couplets or triplets, indicate that
 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672
type 2 IP3R are predominantly located in the ER near the bile
canaliculus while type 1 IP3R are distributed throughout most
regions of the ER with some type 1 IP3R concentrated in ER close
to the plasma membrane in the sinusoidal and canalicular do-
mains [4,28,127]. The results of subcellular fractionation studies
indicate that type 1 IP3R are found in regions of the ER very close
to the plasma membrane, and are held in this location by F-actin
[3840]. Taken together, the results obtained using these different
experimental approaches indicate that a small sub-region of the
ER enriched in type 1 IP3Rs is required for SOC activation.
In an attempt to investigate the role of a putative sub-
component of the ER in the activation of SOCs, H4-IIE cells
expressing aequorin targeted to the cytoplasmic space or ER
were used to measure changes in [Ca2+] in these compartments
under conditions of Ca2+ entry through SOCs. SERCA inhi-
bitors were used to probe the role of SERCAs in the Ca2+ entry
pathway [128]. The results led to the conclusions that the
activation of SOCs and the maintenance of Ca2+ entry through
SOCs is achieved by a small sub-region of the ER Ca2+ store
near the plasma membrane and that SERCAs with a low
sensitivity to inhibition by thapsigargin are required to draw Ca2
+
into this small ER Ca2+ store and to maintain the flow of Ca2+
through SOCs. It was also suggested that diffusion of Ca2+
through the lumen of the ER between the bulk of the ER and the
sub-region near the plasma membrane involved in SOC acti-
vation is relatively slow [128].
7.5. Roles of Gi2, F-actin and PLC1 in facilitating
store-operated Ca2+ channel activation
While it is likely that STIM1 and Orai1 proteins are com-
ponents of the mechanism of activation of liver cell SOCs,
several other proteins appear to be required. Knockdown of
PLC1 in H4-IIE rat liver cells using siRNA was found to be
associated with a substantial decrease in the amplitude of ISOC
initiated by either IP3 or thapsigargin. No interaction between
PLC1 and STIM1 was detected in immunoprecipitation expe-
riments [110]. It was concluded that PLC-1 is required to
couple ER Ca2+ release to the activation of SOCs independently
of any PLC1-mediated generation of IP3 and independently of
a direct interaction between PLC1 and STIM1. These ideas are
similar to those reached in experiments conducted with some
other cell types where it was concluded that a PLC protein is
involved in the activation of receptor-activated Ca2+-permeable
channels and/or SOCs independently of PLC-mediated gene-
ration of IP3 [129131].
ADP-ribosylation of Gi2 by treatment of livers with per-
tussis toxin or the inhibition of Gi2 function using an inhi-
bitory antibody or an inhibitory peptide were each found to
inhibit thapsigargin- and IP3-induced Ca2+ entry (measured
using fura-2) to freshly-isolated rat hepatocytes [132136]
(Fig. 7C). ADP-ribosylation of Gi2 was associated with in-
hibition of the formation of the band of cortical F-actin around
the canaliculus in isolated hepatocyte doublets when spatial
polarity was regained (Fig. 7A), and with some disruption of
the ER (Fig. 7B) [137]. Moreover, studies with hepatocytes,
and some other cell types have shown that Gi2 interacts with
661
F-actin [137]. Disruption of F-actin with cytochalasin D, within
a narrow effective concentration range, inhibited thapsigargin-
and IP3-induced Ca2+ entry [138]. Taken together, these results
indicate that the normal functions of Gi2 and F-actin are
required for the activation of hepatocyte SOCs.
Since the interventions described above inhibited the acti-
vation of SOCs when this was initiated by thapsigargin, as well
as by IP3 and hormones which generate IP3, it was concluded
that the requirements for Gi2 and F-actin are downstream of the
step in which Ca2+ is released from the ER. Thus, it was pro-
posed that Gi2 is not involved in the formation of IP3 catalysed
by PLC, coupled to the vasopressin receptor, and that its role in
the activation of SOCs represents a receptor-independent
function of Gi2 (cf the role of the Gi3 in vesicle trafficking) and
other receptor-independent functions of G-proteins (reviewed in
[139]). The results of experiments conducted with H4-IIE rat
liver cells treated with pertussis toxin suggest that there is no
requirement for Gi2 in the activation of SOCs in this liver cell
line [78]. In rat hepatocytes, the role of Gi2 may be to maintain
hepatocyte spatial polarity since it has been shown that trimeric
G-proteins are involved in determining cell polarity [140]. The
results of other experiments suggest that the normal function of a
monomeric G-protein, possible ARF-1, is also required for the
activation of SOCs in hepatocytes [141].
PLC1, Gi2, a monomeric G-protein and F-actin may play
permissive roles in SOC activation in spatially polarised
hepatocytes (Fig. 6A). The permissive roles of these proteins
may include maintenance of the integrity of the ER and the
putative ER-plasma membrane junctions. One reservation about
this conclusion is that, in the experiments designed to test the
roles of Gi2 and F-actin, the Ca2+ entry pathway involved was
not characterised using electrophysiological techniques.
7.6. Likely physiological functions of store-operated Ca2+
channels in liver cells
In the original concept of capacitative Ca2+ entry developed
by Putney, it was hypothesised that the physiological function
of SOCs is to supply extracellular Ca2+ to refill the ER after Ca2+
is released from this organelle following the actions of PLC-
coupled hormones [142]. This remains a reasonable teleological
hypothesis for the major function of SOCs, although direct
evidence for a role for this pathway under physiological con-
ditions (normal [Ca2+ext]) in hepatocytes and in other non-
excitable cells is limited.
In the absence of Ca2+ext (i.e. in a non-physiological situation),
hormones induce the release of Ca2+ from the ER and this Ca2+,
in turn, is transported out of the cell by the plasma membrane
(Ca2+ + Mg2+)ATP-ase. Re-addition of Ca2+ext leads to refilling
of the ER via SOCs [57,143]. However, it is less clear whether
SOCs play a role in liver cells exposed to hormones under
physiological conditions. The results of experiments which em-
ployed 2-aminoethyl diphenylborate, Gd3+ and SK&F96365 to
inhibit SOCs have provided some evidence that SOCs are re-
quired for the maintenance of hormone-induced Ca2+ oscillations
in hepatocytes [55]. However, interpretation of these results
assumes specificity for the SOC inhibitors employed, and these
662 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672

Fig. 7. Disruption of Gi2 function alters F-actin distribution, disrupts the ER, and inhibits vasopressin-activated Ca2+ entry in rat hepatocytes. (A) Pre-treatment of
livers with pertussis toxin inhibits re-organisation of F-actin in isolated rat hepatocytes which have been plated for 4 h (a, b, pertussis toxin, c, d, control). In freshly-
isolated hepatocytes F-actin (stained using Texas Red-X phalloidin) is principally localised at the cortex (A, a). In control cells plated for 4 h, spatial polarity begins to
be regained and this process is associated with the loss of cortical F-actin and an increase in F-actin around the bile canaliculus (arrow) (A, c). In hepatocytes isolated
from livers treated with pertussis toxin, the re-organisation of F-actin is inhibited (Ab, Ad). (B) Pre-treatment of livers with pertussis toxin induces fragmentation of the
smooth ER in isolated hepatocytes (from [137], with permission). (C) Pre-treatment of livers with pertussis toxin inhibits vasopressin-stimulated Ca2+ entry.
Hepatocytes loaded with fura-2 were incubated in the absence of extracellular Ca2+ and in the presence of vasopressin. Extracellular Ca2+ (1.3 mM) was subsequently
added to initiate Ca2+ entry. (Adapted from [132], with permission.)
 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672
are known to inhibit other types of Ca2+ channels [55]. The
results of other experiments suggest that SOCs are more effective
than at least one type of receptor-activated Ca2+-permeable
channel, the maitotoxin-activated Ca2+ entry pathway, in refilling
the hepatocyte ER [143]. The proposed role of SOCs in hepa-
tocytes in delivering Ca2+ to refill the ER and of (Ca2+ + Mg2+)
ATP-ases in the ER in transporting Ca2+ from the cytoplasmic
space at the mouth of the SOC to the lumen of the ER is analogous
to the roles previously proposed for Ca2+ entry channels and
SERCA pumps in pancreatic acinar cells in re-filing regions of
the ER located next to the acinus with Ca2+ which enters from the
basal region and is moved (tunnelled) through the lumen of the
ER [144].
Another function of SOCs in hepatocytes may be to deliver
Ca2+ to specific locations in the cytoplasmic space near the
plasma membrane, as suggested for other cell types [115,145].
While there is some evidence for such a role for SOCs in some
cell types, for example, in the regulation of adenylate cyclase
activity [145], no direct evidence for this function of SOCs in
hepatocytes has so far been reported. SOCs may also contribute
to transcellular Ca2+ movement. Thus hormone-induced in-
creases in [Ca2+]cyt are accompanied by the extrusion of Ca2+
across the canalicular membrane into bile fluid [146].
The results of experiments conducted using inhibitors of
SOCs suggest that Ca2+ entry through SOCs is required for the
maintenance of bile flow in rats [147]. Recent studies with H4-
IIE rat liver cells and isolated rat hepatocytes employing patch
clamp recording and fura-2 have provided evidence that tauro-
deoxycholic acid and some other choleretic bile acids activate
SOCs, while lithocholic acid and some other cholestatic bile
acids inhibit SOCs. These actions of bile acids are associated with
a re-distribution of STIM1 (Aromataris, E., Castro, J., Rychkov,
G., and Barritt, G.J., unpublished results). Thus SOCs may
mediate some physiological, pathological and pharmacological
actions of bile acids on hepatocytes.
Pharmacological approaches also suggest that Ca2+ entry
through SOCs is required for cell proliferation in human hepa-
toma cells. Thus 2-aminoethyldiphenylborate and carboxyami-
dotriazole inhibited thapsigargin-initiated Ca2+ entry in HepG2
and Huh-7 cells, and this was associated with an inhibition of
cell proliferation [24].
8. Receptor-activated and stretch-activated Ca2+-permeable
channels
Several non-SOC plasma membrane Ca2+-permeable chan-
nels, which can broadly be defined as receptor-activated chan-
nels, have been identified in liver cells and hepatocytes. These are
listed in Table 2, although it should be noted that only a limited
number of species and liver cell lines have so far been studied in
any detail. Receptor-activated Ca2+-permeable channels found in
liver cells include channels activated by intracellular messengers
and/or possibly by proteinprotein interaction. These different
channels were discovered by measuring Ca2+ entry in response to
hormones, specific intracellular messengers (e.g. IP3), toxins
(e.g. maitotoxin) or free radicals. The stretch-activated channels
(also activated by ATP) [148,149] and Ca2+-activated non-
663
selective cation channels activated by ATP, vasopressin and other
agonists [150] have been reasonably well characterised. Ara-
chidonic acid-activated plasma membrane Ca2+-permeable chan-
nels are present in several types of animal cell, and are thought to
be involved in refilling intracellular Ca2+ stores [151]. Arachi-
donic acid-activated Ca2+ entry attributed to the TRPV4 channel
has been described in the HepG2 liver cell line [152]. However,
no arachidonic acid-activated Ca2+-permeable channels could be
detected by patch clamping in H4-IIE rat liver cells or in rat
hepatocytes [99].
The physiological functions of the different types of receptor-
activated Ca2+-permeable channels expressed in hepatocytes are
not well defined. Many admit Na+ as well as Ca2+, and increases
in intracellular Na+ may play important roles in regulating nor-
mal and pathophysiological hepatocyte functions [59,120,150].
Some channels may have quite specific localisations in the
spatially polarised hepatocyte and deliver Ca2+ and Na+ to these
specific regions. Channels with specific activation and inhibition
kinetics may deliver specific types of Ca2+ and Na+ signals
(pulses) to the cytoplasmic space.
9. TRP polypeptides as the molecular counterparts of liver
cell receptor-activated Ca2+-permeable channels
The TRP super family of non-selective cation channels is
comprised of seven sub-families: the TRPC (canonical), the
TRPV (vanilloid), and the TRPM (melastatin) families; the
TRPML (mucolipin) and TRPP (polycysteine) families; and
the TRPA (ankyrin) and TRPN (no mechano receptor
potential C) channels [10,56]. Each TRP channel seems to
have an extraordinary range of cellular functions [10,56]. TRP
channel expression in liver tissue, hepatocytes, and liver cell
lines has been detected using RT-PCR, Northern Blot, in situ
hybridisation, anti-TRP antibodies and functional assays
(Table 3). Some TRP mRNA detected in liver tissue is likely
expressed in non-hepatocyte cell types, such as Kupffer cells
and bile duct epithelial cells. While these results provide
evidence for expression of TRPC1, C3 and M7 in rat hepa-
tocytes, and there is some knowledge of possible functions of
TRPC1 in hepatocytes, and of TRPV4 in HepG2 liver cells,
surprisingly little is known about which other TRP proteins
are expressed in primary hepatocytes and what their cellular
functions are.
Results of immunofluorescence experiments indicate that in
H4-IIE rat liver cells, endogenous TRPC1 is principally located
in intracellular organelles with some expressed at the plasma
membrane [119,120]. Ectopic expression of human TRPC1 in
H4-IIE cells led to a significant enhancement of maitotoxin-
stimulated Ca2+ and Na+ entry compared with the small en-
hancement of thapsigargin-stimulated Ca2+ entry mentioned
above [119]. Suppression of endogenous TRPC1 expression
with trpc1 siRNA led to a small decrease in thapsigargin-sti-
mulated Ca2+ entry and to a larger decrease in maitotoxin- and
ATP-stimulated Ca2+ entry. The suppression of TRPC1 expres-
sion also led to an enhancement of swelling in hypotonic solu-
tion and to enhanced regulated volume decrease [120]. It was
concluded that TRPC1 can be activated by maitotoxin. One
664 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672

Table 2
Receptor-activated Ca2+-permeable non-selective Ca2+ channels detected in the perfused liver, freshly-isolated hepatocytes, hepatocytes in primary culture, and liver
cell lines
Type of Ca2+-permeable
channel
Stretch- and ATP-activated
(chiefly admits Na+)
(16 pS channel)
Cyclic AMP-activated
Maitotoxin-activated
IP3-activated plasma
membrane Ca2+ permeable
channel
Hormone (receptor)-activated
Ca2+-activated non-selective
cation channel
Insulin-activated
Arachidonic acid-activated
Free radical-activated non-
selective cation channel
(16-ps)
Ca2+Mg2+ exchanger
Liver cell preparation Agents used to initiate Technique employed to measure References
Ca2+ inflow Ca2+ inflow
Isolated hepatocytes (rat) Stretch Patch clamp recording [149]
Liver cell line (rat hepatoma) Stretch and ATP Patch clamp recording [148]
Isolated hepatocytes (rat) Glucagon, cyclic AMP Intracellular fluorescent Ca2+ sensor [88,94,186]
analogues, parathyroid hormone
Isolated hepatocytes (rat) Cyclic AMP Northern blot to detect expression of mRNA [165]
and rat liver encoding the CNGC pore-forming subunit
of cyclic nucleotide-gated channel
Isolated hepatocytes (axolotl) Cyclic AMP analogues Intracellular fluorescent Ca2+ sensor [166]
Liver cell line (H4-IIE rat Maitotoxin Intracellular fluorescent Ca2+ sensor [107,119,138]
hepatoma) and isolated (also Mn2+ as substitute for Ca2+) patch
hepatocytes (rat) clamp recording
Isolated hepatocytes IP3 Intracellular fluorescent Ca2+ sensor [8587]
(guinea pig, rat)
Isolated hepatocytes (rat) Vasopressin Intracellular fluorescent Ca2+ sensor [91]
(quenching by Mn2+)
Isolated hepatocytes (rat) Vasopressin Intracellular fluorescent Na+ sensor, [76]
patch clamp recording
Liver cell line ATP, nucleotide analogues Patch clamp recording [150]
(HTC rat hepatoma)
Liver cell line Ca2+ Patch clamp recording [150]
(HTC rat hepatoma)
Liver cell line (HepG2) Ca2+ Patch clamp recording [187]
Isolated hepatocytes (rat) Insulin Intracellular fluorescent Ca2+ sensor [188,189]
Liver cell line Patch clamp recording, intracellular [190]
(HTC rat hepatoma) fluorescent Ca2+ sensor
Liver cell line (HepG2) Arachidonic acid Intracellular fluorescent Ca2+ sensor [152]
Liver cell line (clone 9 cells) Reactive oxygen species Patch clamp recording, intracellular [34]
fluorescent Ca2+ sensor
Hepatocytes and microsomes Isoproterenol [191]
(plasma membrane vesicles) (via cyclic AMP)

possible physiological function of TRPC1 is in the regulation of
hepatocyte volume [119,120].
Studies with HepG2 cells have provided evidence which
suggests the presence of functional TRPV1 channels in this
human liver cell line [152,153]. Thus, capsaicin and RTX,
known activators of TRPV1, were found to stimulate Ca2+
entry (assessed using a fluorescent Ca2+ sensor) and the action
of capsaicin was blocked by the known capsaicin antagonist,
capsazepin. It was also observed that capsaicin induces the
release of Ca2+ from intracellular stores, suggesting that
TRPV1 is located in intracellular membranes and, if appro-
priately activated, can mediate intracellular Ca2+ release.
Incubation of HepG2 cells with hepatocyte growth factor/
scatter factor for 20 h increased capsaicin-stimulated Ca2+
entry in migrating HepG2 cells, leading to the suggestion that
Ca2+ entry through TRPV1 may be involved in the regulation
of liver cell migration [152,153]. While no endogenous
TRPV1 protein could be detected in H4-IIE rat liver cells by
immunofluorescence or by measuring Ca2+ entry in response
to TRPV1 agonists, there is evidence for Ca2+ entry through
TRPV1 in rat hepatocytes (Castro, J., Rychkov, G., and Barritt,
G., unpublished results).
Ca2+ inflow in HepG2 cells was also found to be stimulated
by 4-phorbol-12,13-didecanoate and arachidonic acid, known
activators of TRPV4, suggesting that functional TRPV4 is
expressed in this liver cell line. As mentioned above, a current
attributable to TRPM7 has be been detected in liver cells
(Fig. 8A,B) and this could be suppressed using trpm7 siRNA
(Litjens, T., Rychkov, G., Roberts, M., Chen, J. and Barritt, G.,
unpublished results) (Fig. 8C).
10. Plasma membrane Ca2+-permeable channels activated
by glucagon
Glucagon regulates hepatic carbohydrate, lipid and protein
metabolism, bile flow, and hepatocyte volume and membrane
potential [59,154156]. The results of experiments conducted
using intracellular fluorescent Ca2+ sensors to measure [Ca2+]cyt
indicate that when added alone glucagon or cyclic AMP en-
hance Ca2+ entry to hepatocytes. The magnitude of the en-
hancement varies considerably from one laboratory to another
[94,157159]. The results of other experiments conducted with
the perfused liver and isolated hepatocytes indicate that glu-
cagon can release Ca2+ from intracellular stores in hepatocytes,
 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672 665

Table 3
TRP channels expressed in liver and liver cell lines
TRP Technique employed Species Liver tissue, hepatocytes Comments References
protein for detection or liver cell line
TRPC1 RT-PCR, Western blot, Rat, mouse, AML12, H4-IIE cells, TRPC1 mRNA was detected in all cell [119,120,192194]
immunofluorescence human hepatocytes, liver, types examined, mRNA level in foetal
foetal liver liver is higher than that in liver.
TRPC1 mRNA endogenous TRPC1
protein was detected in H4-IIE cells
with three anti-TRPC1 antibodies
TRPC2 RT-PCR Rat, mouse AML12, H4-IIE Lower mRNA level was detected in [119,192]
cells hepatocytes than that in brain
TRPC3 RT-PCR Rat, mouse, AML12, H4-IIE cells, mRNA level variable among cells types [119,192,194]
human hepatocytes, liver, and higher expression level detected in
foetal liver brain, mRNA not detected in human liver
TRPC5 RT-PCR Human Liver, foetal liver mRNA detected in low expression level [194]
TRPC6 RT-PCR Human Liver, foetal liver mRNA detected in low expression level
TRPC7 RT-PCR Rat, human H4-IIE cells, hepatocytes, mRNA detected in rat liver cells, not [Chen, J L and Barritt, G J
liver and foetal liver detected in human liver tissues unpublished results; 194]
TRPV1 RT-PCR, patch clamp Human Hep G2 cells mRNA detected in low expression level. [152,153,195]
recording and intracellular Capsaicin-activated Ca2+ inflow and
fluorescent Ca2+ sensor TRPV1 current measured. Proposed
role TRPV1 in cell migration.
TRPV2 Dot-blot analysis RT-PCR Rat, human Liver HepG2 cells mRNA detected [152,196]
TRPV3 RT-PCR HepG2 cells mRNA detected [152]
TRPV4 RT-PCR, Northern blot Rat, mouse H4-IIE cells, HepG2 cells, mRNA detected, with abundant [152,197; Chen, J L and
hepatocyte, liver expression in liver cell lines and tissue Barritt, G J unpublished
4-phorbol-12,13-didecanoate- and results]
arachidonic acid-activated Ca2+ inflow
measured
Dot-blot analysis Human, mouse Liver Low expression level [198]
TRPV6 RT-PCR Zebra fish Liver [199]
TRPM4 Northern blot Human Liver Moderate expression level [200]
TRPM5 Northern blot, dot-blot Human Liver Strong signal detected by Northern blot [201,202]
RT-PCR
TRPM7 Northern blot Rat Liver Strong signal detected by Northern blot [103]
In situ hybridisation Zebra fish Liver Abundant expression [102]
Patch clamp recording, Rat H4-IIE cells TRPM7 mRNA (RT-PCR) and TRPM7 [Rychkov, G., Litjens, T.,
RT-PCR current detected Chen, J.L., and Barritt,
G.J. unpublished results]
PKD2 RT-PCR, immunofluorescence Human Liver High expression level [203205]

although the magnitude of observed Ca2+ release also varies
between different laboratories (reviewed in [160]).
The primary pathway of glucagon action involves the G-protein
coupled receptor for glucagon, GS, adenylate cyclase and the
generation of cyclic AMP. Most intracellular effects of cyclic
AMP are mediated by the activation of protein kinase A, but
some are mediated by the binding of cyclic AMP to Epac (ex-
change protein directly activated by cyclic AMP) and cyclic
nucleotide-gated cation channels [156,161]. Glucagon also in-
creases IP3 concentrations in hepatocytes through coupling of the
glucagon receptor to Gq and PLC [162164]. Thus the glucagon
receptor can interact with two trimeric G-proteins, Gs and Gq. In
hepatocytes increases in IP3 induced by glucagon are substan-
tially lower than those induced by vasopressin and epinephrine,
the receptors for which are predominantly coupled to Gq and
PLC [162164].
The results of recent studies using patch clamp recording
with rat hepatocytes have shown that glucagon activates a small
inwardly rectifying Ca2+ current with characteristics similar to
those of the Ca2+-selective hepatocyte SOC, and as discussed
above, a larger outwardly rectifying Cl current similar to that
activated by cell swelling [9]. Evidence was presented to
indicate that these effects of glucagon involve IP3, cyclic AMP
and Epac, but do not involve protein kinase A.
Another Ca2+-permeable channel in the hepatocyte plasma
membrane which has been suggested as a possible target of
glucagon and cyclic AMP is the photoreceptor cyclic nucleo-
tide-gated non-selective cation channel [165]. While there is
evidence that mRNA encoding this channel is expressed in liver
[165], no convincing evidence for the presence of functional
cyclic nucleotide-gated Ca2+ permeable channels in mammalian
hepatocytes has so far been reported. It is possible, though, that
a cyclic nucleotide-gated non-selective cation channel accounts
for the observed cyclic AMP-activated Ca2+ entry in axolotl
hepatocytes which exhibit a substantial and convincing cyclic
AMP-activated Ca2+ entry [166].
Glucagon acts synergistically with vasopressin, epinephrine,
phenylephrine, and other G-protein coupled receptors which
666 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672

Fig. 8. Currents attributable to TRPM7 in H4-IIE rat liver cells. (A) Currenttime plot showing development of an outward current, measured at 100 mV, due to washout
of intracellular Mg2+. (B) Currentvoltage plots recorded at the time points indicated in panel A, immediately after achieving whole cell configuration (1), and after the
TRPM7 current has fully developed (2). (C) Averaged currentvoltage plots obtained 200 s after achieving whole cell configuration, for cells in which TRPM7
expression is ablated by trpm7 siRNA, and for control cells transfected with control siRNA. Traces in B and C are recorded in response to 100 ms voltage ramps ranging
from 138 to 102 mV. To activate TRPM7 currents, Mg2+ was omitted from the pipette solution and intracellular Ca2+ was buffered to 100 nM by EGTA.

induce IP3 formation and Ca2+ release from intracellular stores,
to potentiate Ca2+ entry [157,167171]. The degree of poten-
tiation varies somewhat between liver and hepatocyte prepara-
tions in different laboratories. The synergistic or potentiating
action of glucagon is mediated by cyclic AMP, since this intra-
cellular messenger can substitute for glucagon [160]. Synergy
involves the release of Ca2+ from the ER, since it has been
shown that glucagon or cyclic AMP enhance Ca2+ entry when
this is initiated by SERCA inhibitors such as DBHQ [94]. The
Ca2+ permeable channel which is synergistically activated has
not been characterised by electrophysiological studies, but is
most likely the liver cell Ca2+-selective SOC.
The mechanism(s) by which cyclic AMP enhances IP3- or
SERCA inhibitor-initiated Ca2+ entry has not been elucidated, but
several possibilities have been suggested. Cyclic AMP acting
through protein kinase A may enhance Ca2+ uptake by mito-
chondria located near the plasma membrane which, in turn, may
reduce feedback inhibition by Ca2+ of SOCs [167]. Cyclic AMP
may also enhance the formation of IP3 and/or increases the
sensitivity of IP3 receptors to IP3 and hence, potentiate the release
of Ca2+ from the ER and the subsequent activation of SOCs [160].
It has also been shown that cyclic GMP potentiates the
synergistic action of glucagon and vasopressin on Ca2+ entry in
rat liver, and in guinea pig hepatocytes potentiates the release of
Ca2+ from the ER mediated by IP3 [172,173]. The mechanism
of action of cyclic GMP on Ca2+ entry appears to be complex
but may include enhancement of Ca2+ release from the ER and a
subsequent enhanced activation of SOCs.
11. Hepatocyte plasma membrane Ca2+-permeable
channels activated in response to ischaemia reperfusion
injury of the liver
When livers are subjected to ischaemic reperfusion injury, an
increase in total hepatocyte Ca2+ and in the amount of Ca2+ in
mitochondria is observed immediately following the onset of
reperfusion [174177]. Studies with isolated hepatocytes sub-
jected to hypoxia or anoxia show a sustained increase in [Ca2+]cyt
upon re-oxygenation [178180]. This is due to enhanced plasma
membrane Ca2+ entry as well as release of Ca2+ from intracellular
stores [179,180].
Reactive oxygen species generated by Kupffer cells and
hepatocytes during the initial stages of reperfusion are thought to
be one of the main mediators of enhanced Ca2+ entry to
hepatocytes (reviewed in [181]). NO and reactive nitrogen
species, generated at later stages of reperfusion, may also affect
Ca2+ entry [181]. While the Ca2+ permeable channels involved
in enhanced Ca2+ entry to hepatocytes in ischemia reperfusion
injury have not been clearly identified, the results of experiments
conducted with a liver cell line have shown that reactive oxygen
species can activate a 16 pS Ca2+-permeable non-selective
cation channel [34] (Table 2). TRPM7 and several other TRP
channels are known to be activated by reactive oxygen species
[182,183] and NO [184]. In response to toxic insults which
induce the generation of reactive oxygen species, TRPM7 me-
diates Ca2+ and Na+ entry to neurons which, in turn, leads to
cell death [182]. TRPM7 is expressed in liver cells (Fig. 8 and
Table 3) and is a possible candidate for mediating Ca2+ entry in
hepatocyte death initiated by reactive oxygen species.
12. Conclusions
The following conclusions can be reached from the above
discussions of liver cell plasma membrane Ca2+-permeable
channels. The spatial and temporal organisation of Ca2+ entry
through Ca2+-permeable channels in the hepatocyte plasma
membrane are critical in generating specific cytoplasmic Ca2+
signals. The liver cell Ca2+-selective SOC, with characteristics
similar to those of the CRAC channels in lymphocytes and mast
cells most likely plays a major role in Ca2+ entry to liver cells.
This is complemented by some receptor-activated Ca2+-perme-
able channels, at least one type of ligand-gated Ca2+ permeable
channel (the P2X purinergic-activated channel) and stretch-
activated Ca2+-permeable channels. Several receptor-activated
Ca2+ channels have been identified and partially (often in-
completely) characterised. Some TRP Ca 2+ - and Na + -
 G.J. Barritt et al. / Biochimica et Biophysica Acta 1783 (2008) 651672
permeable channels are known to be expressed in liver cells.
While TRP polypeptides are unlikely to be the molecular
components of SOCs, they are likely to comprise many of the
liver cell receptor-activated and stretch-activated Ca2+-perme-
able channels. Further experiments are required to characterise
liver cell receptor-activated Ca2+ permeable channels more
completely, and to determine the molecular nature, mechanisms
of activation, and precise physiological function of all
hepatocyte plasma membrane Ca2+ permeable channels. It
will be important that experiments directed towards further
elucidation of the molecular properties and physiological
functions of Ca2+ permeable channels be conducted as far as
possible with primary hepatocytes since the spatial polarity and
differentiation of these cells is critical to the intracellular Ca2+
signals.
Acknowledgements
The authors gratefully acknowledge the assistance of Karen
Jennings in preparation of the manuscript. Research conducted
in the authors' laboratories which has contributed to this review
is supported by grants from the National Health and Medical
Research Council of Australia, the Australian Research Council,
and the Flinders Medical Centre Foundation of South Australia.
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