Chloride channels: a molecular perspective
Thomas J Jentsch
Plasma membrane Ct- channels perform a variety of functions, including control of excitability in neurons and muscle, cell volume regulation and transepithelial transport. Structurally, three classes of Cl- channels have been
identified: ligand-gated, postsynaptic Cl- channels (e.g. GABA and glycine receptors); the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels (which belong to the traffic ATPase superfamily); and the CLC family of Cl- channels. Recent developments of note include further characterization of the expanding CLC Cl- channel family, advances in understanding the regulation of the CFTR Cl- channel and its emergent role as a regulator of other channels, clarification of issues related to swelling-activated Cl- channels, and the discovery that several co-transporter molecules are now known to induce Cl- currents in Xenopus oocytes.
Address
Centre for Molecular Neurobiology Hamburg (ZMNH),
Hamburg University, Martinistrasse 52, O-20246 Hamburg, Germany Abbreviations
CFTR cystic fibrosis transmembrane conductance regulator GABA yaminobutyric acid
NBF nucleotide-binding fold
Current Opinion in Neurobiology 1996, 6:303-310
0 Current Biology Ltd ISSN 0959-4388
Introduction
Chloride channels are found in the plasma membrane of probably all eukaryotic cells. In contrast to the intracellular concentration of ions such as Caz+ or Na+, the intracellular concentration of Cl- is close to its electrochemical equilibrium. Thus, Cl- has no established function as a second messenger, and is only rarely involved in electrical excitation, Rather, in a way similar to K+ channels, Cl- channels dampen electrical excitability. This is most evident in the nervous system, where GABA and glycine receptors generally generate inhibitory postsynaptic currents, and in skeletal muscle. Mutations in glycine receptor subunits result in hyperekplexia (startle disease) [l], which is attributable to a lack of inhibitory input in a subset of neurons. Furthermore, mutations in the skeletal muscle Cl- channel protein CLC-1 lead to muscle hyperexcitability, resulting in myotonia [2,3]. Interestingly, GABA receptors may have, as an exception, excitatory effects in certain neurons where co-transporters can raise the intracellular Cl- concentration above its electrochemical equilibrium [4].
Chloride is also important for the osmotic control of water flow. Although Cl- is accompanied by K+ when its efflux via swelling-activated Cl- channels counteracts osmotic swelling, Cl- is often joined by Na+ when it creates tiny transepithelial osmotic gradients necessary for fluid secretion or absorption. Being not far from its electrochemical equilibrium, transport of Cl- across both membranes (apical and basolateral) is energetically cheap. The importance of Cl- channels for transepithelial transport is apparent from disorders such as cholera or cystic fibrosis, which are attributable to an activation or mutational inactivation, respectively, of CAMP-regulated Cl- channels. Further, the recent discovery that mutations in a kidney Cl- channel (CLC-5) lead to kidney stones [S”] again suggests a defect in transepithelial transport.
Cl- channels also fulfil important roles in intracellular organelles, including endocytotic and synaptic vesicles. This topic has been reviewed recently f6] and will not be covered here. Instead, this review will focus primarily on Cl- channels whose molecular identity is known, without, however, considering postsynaptic GABA and glycine receptors. The emphasis is placed on the most recent developments in this field. Focusing exclusively on Cl- channels in the nervous system would ignore many important developments, and is thus avoided.
CLC chloride channel proteins
The CLC proteins form the only known large gene family of Cl- channels. They show a high degree of evolutionary conservation and homologues are present in yeast and bacteria. CLC proteins comprise about 12 transmembrane domains (Figure lb) 17,801 and function as multimers [2,9].
304 Signalling mechanisms
Figure 1
(a) GABA and glycine receptors (b) CLC Cl’ channels
D9.. ... D12 ... ...
(c) CFTR
Q 1996 Current Opinion in Neurobiology
Topology models for GABA and glycine receptors, CLC Cl- channels, and CFTR. (a) GABA and glycine receptors are typical members of the ligand-gated ion channel superfamily, which includes the nicotinic acetylcholine receptor. Members have four transmembrane domains (Ml-M4) and probably function ss (mostly hetero-) pentamers. (b) CLC Cl- channels have about 12 tran~embrane domains (01 -Dl2), the exact topology of which is not yet known [8*,29]. A previous model I1 01 had to be modified, as the loop between D8 and 09 is now known to be glycosylated [9,13]. Analysis of new CLC genes suggests that D4 is not a conserved hydrophobic domain, eliminating it as a transmembrane span [20]. Whereas D2 is thought not to cross the membrane in an alternative model [91, several algorithms predict that it is a very good candidate for a transmembrane domain. Further, deletion of this domain destroys channel activity [5**,7] and results in kidney stones [5**]. The topology in the broad hydrophobic region OS-D1 2 is obscure, and the conserved region 013 is known to be cytoplasmic [l 11. (c) CFTR has
two blocks of six putative transmembrane domains (TM1 -TM6 and TM7-TM12), which are followed by the nucleotide-binding folds NBFl and NBFP, respectively, The regulatory ‘R’ domain must be phosphorylated before regulation of channel opening by ATP (at the NBFs) can occur.
detail exciting developments regarding specific CLC Cl- mechanism by which a defect in a Cl- channel leads to
channel proteins. hypercalciuria and proteinuria is still unclear.
CLC-5 CLC-1
Dendrogram showing the degree of similarity between the known mammalian members of the CLC superfamily. Members of the fmt branch, which includes CLC-1, CLC-2, CLC-Ka and CLCKb, are those most closely related to the Torpedo channel UC-O, the founding member of this gene family [lo]. The second branch, including CLC-3, CLC-4 and CLC-5, contains those members most closely related to the yeast homologue SCCLC 1301. (Interestingly, the recent completion of the yeast genome project indicates that there is only one CLC gene in yeast, which we suggest calling ‘scCW.) Finally, a recent add&ion to the CLC sup&amity is the CLC-6 and CLG7 branch. Tissue expression patterns and proposed functions are also indicated.
Thus, it seems unlikely that all these mutations affect gate structures directly.
CLC-0
Whereas many cation channels sense the membrane voltage using charged amino acids in transmembrane domains, ‘fast’ gating of CLC-0 can be described by a fundamentally different model in which the permeating anion itself provides the ‘gating charge’ [@I. Opening rates are increased by the presence of anions close to the internal end of the pore. This model provides a simple explanation for the observed voltage and Cl--concentration dependence of CLC-0 gating, and is supported by the analysis of mutations at the very end of the transmembrane spans. These mutations modulate gating, single-channel conductance, and ion selectivity, suggesting that this region is important for permeation. Ion selectivity is however affected by mutations in other regions as well [Z], so it has not yet been possible to convincingly identify all the structures directly lining the pore region.
CLC-2
CLC-2 can be activated by cell swelling or strong hyperpolarization in Xenopus oocytes [l I]. Superficially similar currents are present in native cells, including epithelia [23-251, neurons [26,27] and osteoblasts [28], but
the osmosensitivity has often not been checked and may even be reversed [28]. Although northern analysis suggests that CLC-2 is expressed ubiquitously, in situ hybridization reveals that CLC-2 is expressed differentiaIly in brain 1271, according to a pattern that differs from that of CLC3 [15,17]. It was proposed that CLC-2 serves to prevent neuronal Cl- accumulation above equilibrium, thereby modulating the effects of postsynaptic GABA receptors [4,26]. Consistent with this hypothesis, expression was observed to be absent or low in neurons exhibiting a ‘paradoxical’ excitation by GABA, attributable to a Cl- efflux via GABA receptors in cells with a high internal Cl- concentration [27].
CLC-4, CLC-6, CLC-7 and CLC-KS
306
Signalling
mechanisms
domain, and as a similar deletion in CLC-5 eliminates
channel activity and leads to kidney stones [5"'].
T h e lack of functional expression could be explained by
complicated regulation of channel activity or by the lack
of important subunits, among others. Alternatively, these
putative channels might serve intracellular roles. T h e
iron-repressible
petite
phenotype (deficiency in respiration)
of a yeast strain deleted for the yeast C L C homologue
scCLC encoded by the
GEF-1
gene ([30]; see also
Figure 2 legend) may point in the direction of the latter
explanation.
Multiple functions of CFTR
An increasing number of reports indicate that C F T R ,
the gene product defective in cystic fibrosis, performs
regulatory roles in addition to being a cAMP-activated
C I - c h a n n e l . This additional function has been signifi-
cantly, albeit indirectly, bolstered by the discovery that
the sulfonylurea receptor (SUR) regulates a m e m b e r of
the inwardly rectifying K ÷ channel family (BIR) to yield
ATP-semitive K ÷ channels [31]. Both C F T R and the
sulfonylurea receptor belong to the superfamily of traffic
ATPases, combining two blocks of transmembrane spans
with two nucleotide-binding folds (Figure lc), although
their topologies differ in detail.
Data obtained previously indicated that C F T R regulates
an outwardly rectifying CI- channel expressed in many
cells [32]. Intriguing experiments now suggest that this
regulation is mediated by ATP, which leaves the cell
in response to C F T R stimulation [33°,34]. This exodus
might be achieved via a pathway closely regulated by
C F T R , or directly via the C F T R pore itself, as suggested
previously [35]. On the other hand, no effect of C F T R on
ATP efflux was found in a different system [36]. A direct
ATP-channel function of C F T R may be surprising for a
pore otherwise highly selective for small anions. Indeed,
several groups have reported recently that C F T R does
not conduct ATP [37]. Extracellular ATP may activate
outwardly rectifying CI- channels by interacting with
purinergic receptors [33°,38] Whether this activation is
mediated by direct interaction, intracellular Ca 2÷, and/or
other pathways is not yet clear, and the molecular identity
of the outward rectifier is unknown. Intriguingly, some
mutations associated with cystic fibrosis have been shown
to have differential effects on intrinsic C I - channel and
regulator activities [39].
In addition to a defective CI- conductance, airway
epithelia of cystic fibrosis patients show an increase in
Na÷ currents. It is now known that C F T R leads to
cAMP-stimulated inhibition of co-transfected epithelial
Na ÷ channels [40"]. Although this effect is small in
fibroblasts, the inhibition observed in epithelial cells
seems substantial. Interactions between both channels
were also observed in the
Xenopus
oocyte expression
system [41]. T h e mechanism of this interaction is still
unclear, but these observations suggest that a search for
interactions with other channels might be fruitful. Indeed,
first reports indicate that C F T R may also regulate inwardly
rectifying K ÷ channels (CM McNicholas, WB Guggino,
SC Hebert, ME Egan,
Pediatr Pulmonol
1995, 19:77), and
indirect evidence suggests that C F T R may also affect
volume-activated K ÷ channels [42]. Clearly, more research
is needed to elucidate these effects, as it would be
surprising if C F T R were involved in all these diverse
processes.
In view of these regulatory roles, the additional function
of C F T R as a C I - channel is surprising, but well
established. T h e ATP-channel model [33"], although very
controversial [36,37], reconciles both functions. As C F T R
belongs to the traffic ATPase family, several studies
have focused on the roles of its two nucleotide-binding
folds (NBFs) [43",44"°,45,46]. In contrast with 'normal'
transport ATPases, in which ATP is hydrolyzed upon each
active transport cycle, the passive pore of the C F T R
channel is thought to be regulated by ATP hydrolysis.
This regulation can only occur after C F T R has been
phosphorylated by protein kinase A at the 'R domain'.
Extending previous results [47], the new data suggest
that ATP hydrolysis at NBF1 is important for channel
opening. Binding of ATP at the second N B F (NBF2) is
thought to stabilize the open state, and ATP hydrolysis
at NBF2 facilitates channel closure [44"%46]. A previously
undetected second open state (02) with a slightly different
conductance has been exploited in an elegant series
of experiments
[44"].
T h e results suggest that ATP
hydrolysis at NBF2 leads to a transition from the first
open state (O1) to O 2, from which the channel can
close spontaneously [44"']. Although these studies only
indirectly suggest that ATP is hydrolyzed by C F T R , new
biochemical data indicate that NBF1 (in a fusion protein)
may indeed function as an ATPase [48].
T h e most frequent mutant form of C F T R , AF508, does
not reach the cell surface but instead is degraded in
a pre-Golgi compartment. It has now been shown that,
rather like other m e m b r a n e proteins, both wild-type and
mutant C F T R proteins are degraded in part by the
ubiquitin-proteasome pathway [49,50].
Although changes in ion selectivity have been corre-
lated with mutations in several transmembrane domains
([51,52]; but, see [53]), the pore of this extensively studied
molecule is not yet well understood. It is a sure bet that
C F T R still has many surprises in store.
Chloride channels and regulation of cell
volume
(MDR1) generates volume-activated C I - c h a n n e l s [54]
has stirred considerable interest and controversy over
the years [55-60]. It now seems clear that M D R 1
is not itself a CI- channel. Instead, it is thought to
confer regulation by protein kinase C onto endogenous
swelling-activated CI- channels [61]. As MDR1 is a
traffic ATPase (indeed, a typical one), this suggestion
fits very well the regulatory roles of C F T R and the
sulfonylurea receptor. (Interestingly, but also controver-
sially, M D R l - - l i k e C F T R - - h a s been suggested to be
an ATP channel [62].) T h e mechanism of interaction
between MDR1 and the volume-activated channel is
nonetheless not known, and the regulatory role has been
questioned also [63].
Osmotic swelling activates C L C - 2 in the oocyte expression
system, and structures important for this regulation have
been localized to its amino terminus [11]. CLC-2 does not
however underlie the typical volume-activated CI- channel
observed in numerous cell types. In contrast with CLC-2,
the typical volume-activated current displays an I->CI-
permselectivity, is outwardly rectifying, deactivates at
positive potentials, and depends on intracellular ATP. A
detailed study in glioma cells [64 °] has shown that this
channel has a single-channel conductance of 15-50pS
(depending on voltage), resolving a discrepancy between
single-channel [65] and noise [66,67] analysis, which
suggested a lower value. This current is blocked by
extracellular nucleotides in several cell types [68-70],
resembling the current seen upon overexpression of
plcln, a putative C I - c h a n n e l [71]. Indeed, antisense
oligonucleotides [70] or antibodies [72] directed against
plc1 n inhibit the volume-activated CI- current in native
cells. A convincing proof that plci n and the CI- channel
are one and the same is missing, however, and it is
possible that plcI n instead interacts with endogenous
CI- channels [72].
In the case of regulatory volume decrease, a loss of
intracellular organic osmolytes (taurine and
myo-inositol,
among others) may be equally important as a loss of KCI.
An increasing body of evidence suggests that this loss may
involve the same swelling-activated anion channel [73-75].
C h l o r i d e c u r r e n t s a s s o c i a t e d w i t h
c o - t r a n s p o r t e r s
Several co-transport or counter-transport molecules elicit
anion currents when expressed in
Xenopus
oocytes. For
instance, several members of an excitatory amino-acid/Na ÷
co-transporter family were shown to yield aspartate-
evoked CI- currents when expressed in
Xenopus
oocytes
[76,77]. T h e s e rather small currents were not stoichio-
metrically coupled to amino acid transport, suggesting
that these transporters may have an additional function
of a 'ligand-gated' CI- channel. This could dampen cell
excitability upon neurotransmitter re-uptake.
In addition, expression of a renal sodium-phosphate
co-transporter (NaPi-1) in
Xenopus
oocytes yielded quite
large, time-independent, slightly outwardly rectifying
anion currents with an I-->CI- selectivity [78], which
superficially resemble the currents reported, for example,
for CLC-3 [15]. This current was not directly d e p e n d e n t
on the substrates of the co-transporter (i.e. Na ÷ and
PO42-).
Finally, expression in
Xenopus
oocytes of the trout
erythrocyte anion exchanger (AE, or band 3), but not of
mouse anion exchanger AE1, induced increased taurine
fluxes and a sizeable CI-conductance that was only slightly
outwardly rectifying [75]. Analysis of chimeric transporters
has shown that an extracellular loop and carboxy-terminal
transmembrane domains are necessary for the induction of
these currents.
It has not yet been established whether these proteins pos-
sess dual transporter/channel activities, or whether their
expression in
Xenopus
oocytes activates endogenous chan-
nels. Oocytes possess a variety of endogenous CI- chan-
nels, w h i c h - - i n some cases, very r e p r o d u c i b l y - - m a y be
activated by the overexpression of foreign proteins that
are not themselves channels. Thus, expression of diverse
membrane proteins elicits hyperpolarization-activated an-
ion currents in
Xenopus
oocytes [20,79°,80]. T h e s e currents
were previously thought to be specific for phospholemman
[81]. Several criteria must be m e t before a cloned c D N A
can safely be assumed to encode a channel [29]. One
can, however, by no means exclude the possibility that
molecules have dual transporter/channel functions. This
scenario seems particularly likely for the aspartate-gated
CI- currents.
C o n c l u s i o n s
T h o u g h three gene families of CI- channels are known,
our molecular picture of CI- channels is far from complete.
Several important channels have not yet been cloned,
including the seemingly ubiquitous volume-activated
CI- channel (which may also conduct organic osmolytes),
and Ci- channels activated by intracellular CaZ+. Homol-
ogy cloning continues to uncover new m e m b e r s of the
C L C family, but many of these proteins have yet to
be assigned a function. In the absence of unambiguous
functional expression, it is even possible that some are
not even CI- channels. Many proteins overexpressed in
308 Signalling mechanisms
In addition to the well recognized roles of postsynaptic
ligand-gated CI- channels for signal transduction in
the CNS, the role of voltage-activated C I - c h a n n e l s
in controlling electrical excitability becomes increasingly
clear. This is most obvious for skeletal muscle [2], but
probably is also true for neuronal cells [26,27], and it
would not be surprising if genetic defects in brain CI-
channels turn out to be the basis for certain inherited
neurological disorders. T h e emerging complex regulations
of ion channels, as exemplified by the multiple functions
of C F T R , is likely to add an additional level of complexity
to neurobiology as well.
Acknowledgements
l thank Michael Pusch, Blanche Schwappach, and Klaus Steinmeyer for critical reading of the manuscript. Work in the author's laboratory is supported by the Deutsche Forschungsgemeinschaft, the US Muscular Dystrophy Association, and the Fonds der Chemischen lndustrie.
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• of special interest • • of outstanding interest
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This important work investigates the effects of CFTR on the epithelial Na + channel ENaC in cells transfected with all three ENaC subunits. Using epithelial cell layers of transfected MDCK cells in CI--free solutions, it is shown that forskolin (which raises cAMP) stimulates short-circuit current in the absence of CFTR. This current is mediated in large part by ENaC in the apical membrane. In cells co-transfected with CFTR, in contrast, cAMP causes a slight inhibition. Although these effects are large, whole-cell cur- rent measurements in transfected 3T3 cells yield similar, but much less im- pressive results. Broadly similar results have been obtained in the Xenopus
oocyte expression system [41 ]. The mechanism of interaction between both channel molecules is still unclear. The results of this work are highly relevant for cystic fibrosis, as an increase in Na + re-absorption contributes to lung disease in the patients studied.
41. Mall M, Hipper A, Greger R, Kunzelmann K: Wild type but not ~F508 CFTR Inhibits Na + conductance when coexpressed in
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42. Valverde MA, O'Brien JA, Sep01veda FV, Ratctiff RA, Evans M J, Colledge WH: Impaired cell volume regulation in intestinal crypt epithelia of cystic fibrosis mice. Proc Nat/Acad Sci USA 1995, 92:9038-9041.
43. Carson MR, Travis SM, Welsh M J: The two nucleotide-blndlng • domains of cystic fibrosis transmombrane conductance
regulator (CFTR) have distinct functions in controlling channel activity. J Bio/Chem 1995, 270:1711-1717.
Using patch-clamp analysis of transfected cells, the analysis of a number of CFTR point mutants, and of ATP and non-hydrolyzable analogs indi- rectly, suggests that ATP hydrolysis at NBF1 initiates burst activity (during which the channel fluctuates many times between open and closed states), whereas hydrolysis of ATP at NBF2 terminates the burst. Non-hydrolyzable ATP analogs prolong the duration of only some burets. These conclusions are broadly similar to those presented in [44"].
44. Gunderson KL, Kopito RR: Conformational states of CFTR • . associated with channel gating: the role of ATP binding and
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Heavy electrical filtering (10 Hz) allows the detection of a previously unknown open state (O 2) of CFTR reconstituted into lipid bilayere. 02 differs by only 1 pS from the other open state O1. Statistical analysis shows that channel gating predominantly occurs in an asymmetric cycle C->O!--~O2--)C, where C is the closed state. This indicates an input of energy, which is most likely supplied by ATP hydrolysis. Non-hydrolyzable compounds lock CFTR in the O1 state. The predominant irreversible step is the O1--)O 2 transition, which is coupled to ATP hydrolysis at NBF2. These conclusions are supported by the analysis of point mutants. Thus, this elegant study suggests that each cycle of CFTR opening is coupled to the hydmlysia of one ATP at NBF2 (and possibly one ATP at NBF1, the role of which is less clear). The appar- ent contrast with a study on intact cells [43"] - which suggests that ATP hydrolysis at NBF2 terminates a burst of activity - may be attributable, in part, to the heavy filtering employed here, which may not allow the detection of fast closures.
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46. Wilkinson D J, Mansoure MK, Watson PY, Smit LS, Collins FS, Dawson DC: CFTR: the nucleotide binding folds regulate the accessibility of the activated stste. J Gen Physiol 1996, 107:103-119.
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48. Ko YH, Pedersen PL: The first nucleoflde binding fold of the cystic fibrosis tmnsmembrane conductance regulator can function as an active ATPase. J Bio/Chem 1995, 270:22093-22096.
49. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, Riordan JR: Multiple proteolytic systems, Including the proteasome, contribute to CFTR processing. Ceil 1995, 83:129-135.
50. Ward LW, Omura S, Kopito RR: Degradation of CFTR by the ubiqultln-proteasome pathway. Ceil 1995, 83:121-127. 51. Anderson MP, Gregory R J, Thompson S, Souza DW, Paul S,
Mulligan RC, Smith AE, Welsh M J: Demonstration that CFTR Is a chlodde channel by alteration of its anion selectivity. Science 1991, 253:202-205.
52. McDonough S, Davidson N, Lester HA, McCarty NA: Novel pore- lining residues In CFTR that govem permeation and open- channel block. Neuron 1994, 13:623-634.
53. Carroll TP, Morales MM, Fulmer SB, Allen SS, Rotte TR,
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55. Mori XK, Bond TD, Loo TW, Clarke DM, Bear CE: Failure of P-glycoprotein (MDR1) expressed in Xenopus oocytes to produce swelling-activated chlodde channel activity. J Physiol (Lond) 1995, 468.3:707-714.
56. Rasola A, Galietta I_IV, Gruanert DC, Romeo G: Volume-sensitive chloride currents in four epithelial cell lines are not directly correlated to the expression of the MDR-1 gene. J Biol Chem
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71. Paulmichl M, Li Y, Wickman K, Ackerman M, Peralta E, Clapham DE: New mammalian chloride channel identified by
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74. Roy G: Amino acid current through anion channels in cultured human glial cells. J Membr Bio11995, 147:35-44.
75. Fi6vet B, Gabillat N, Borgese F, Motels R: Expression of band 3 anion exchanger induces chloride current and taurine transport: structure-function analysis. EMBO J 1995, 14:5158-5169.
76. Fairman WA, Vandenberg RJ, Arriza JL, Kavanaugh MP, Amara SG: An excitatory amino-acid transporter with properties of e ligand-gated chloride channel. Nature 1995, 375:599-603. 77. Wadiche JI, Amara SG, Kavanaugh MP: Ion fluxes associated
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78. Busch AE, Schuster A, Waldegger S, Wagner CA, Zempel G, Broer S, Biber J, Murer H, Lang F: Expression of s renal type I sodium/phosphate-transporter (NaPI-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions. Proc Natl Acad Sci USA 1 g96, in press. 79. Tzounopoulos T, Maylie J, Adeiman JP: Induction of endogenous • channels by high levels of heterologous membrane proteins in
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This paper shows that overexpression of many membrane proteins, including those that do not function as channels, induces slowly hyperpolarization- activated currents in Xenopus oocytes. Thus, these currents, which were thought to be specific for phospholemman [81], are attributable to an acti- vation of an endogenous Xenopua oocyte channel. Similar observations have been made for non-functional CLC proteins [2,20]. The experiments strongly suggests that hyperpolarization opens a non-specific cation channel, and that the inflow of Ca 2+ activates a Ca2+-dependent CI- channel. Thus, this paper investigates one of the pitfalls encountered when studying functional expression of CI- channels.
80. Shimbo K, Brassard DL, Lamb RA, Pinto LH: Viral and cellular small integral membrane proteins can modify ion channel endogenous to Xenopus oocytes. Biophys J 1995, 69:1819-1829.
81. Moorman JR, Palmer C J, John JE, Durieux ME, Jones LR: Phospholemman expression induces a hyperpolsrlzatlon- activated chloride current in Xenopus oocytes. J Biol Chem 1992, 267:14551-14554.