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PubMed1997

Molecular identification of a volume-regulated chloride channel.

A volume-regulated chloride current (ICl.vol) is ubiquitously present in mammalian cells, and is required for the regulation of electrical activity, cell volume, intracellular pH, immunological responses, cell proliferation and differentiation. However, the molecule responsible for ICl.vol has yet to be determined. Although three putative chloride channel proteins expressed from cloned genes (P-glycoprotein, pICln and ClC-2 ) have been proposed to be the molecular equivalent of ICl.vol, neither P-glycoprotein nor pICln is thought to be a chloride channel or part thereof, and the properties of expressed ClC-2 channels differ from native ICl.vol. Here we report that functional expression in NIH/3T3 cells of a cardiac clone of another member of the ClC family, ClC-3, results in a large basally active chloride conductance, which is strongly modulated by cell volume and exhibits many properties identical to those of ICl.vol in native cells. A mutation of asparagine to lysine at position 579 at the end of the transmembrane domains of ClC-3 abolishes the outward rectification and changes the anion selectivity from I- > Cl- to Cl- > I- but leaves swelling activation intact. Because ClC-3 is a channel protein belonging to a large gene family of chloride channels, these results indicate that ClC-3 encodes ICl.vol in many native mammalian cells.

3T3 Cells↗

PubMed1997

Osmosensitivity of the hyperpolarization-activated chloride current in human intestinal T84 cells.

The osmosensitivity of the hyperpolarization-activated chloride current (I(Clhyp)) in T84 cells was studied using the whole cell patch-clamp recording configuration. Hypotonicity is known to activate an outwardly rectifying chloride current (HIORC) distinct from I(Clhyp) in these cells. The differing sensitivities of HIORC and I(Clhyp) toward inhibitors (1,9-dideoxyforskolin blocked HIORC but not I(Clhyp), and Cd2+ inhibited I(Clhyp) but not HIORC) allowed us to investigate the osmoregulation of I(Clhyp). Hypotonicity induced an increase in I(Clhyp) amplitude. Protein phosphatase inhibitors prevented this effect, and hypotonic solutions became slightly inhibitory. Hypertonicity resulted in a transient increase in I(Clhyp) amplitude followed by a large decrease. The complex responses of I(Clhyp) to osmotic changes indicate that these signals affect the same channel via multiple transduction pathways. The responses of I(Clhyp) to hypotonicity have features in common with the responses of ClC-2 channels expressed in Xenopus oocytes (activation) and with hyperpolarization-activated chloride currents in other cell types, such as osteoblasts and mandibular duct cells (inhibition).

Cells, Cultured↗

PubMed1997

Inhibition of the inward-rectifying Cl- channel in rat choroid plexus by a decrease in extracellular pH.

1. The sensitivity of the inward-rectifying Cl- channel in choroid plexus to changes in external pH (pHo) was examined. 2. Cl- currents were recorded using whole-cell patch-clamp methods. The inward-rectifying channel was activated by 375 nM of the catalytic subunit of protein kinase A which was added to the electrode solution. 3. Reducing pHo from 7.3 to 6.5 inhibited the inward-rectifying Cl- currents, whereas an increase in current was observed when pHo was elevated to 8.5. The inhibition of the conductance exhibited a sigmoidal relationship with decreasing pH over a range of 8.5 to 5.5. A half-maximal inhibition of the current was observed at pH 7.3. 4. The inhibition of the whole-cell current by reducing pHo suggests that it is carried by channels which are distinct from other inward-rectifier Cl- channels, e.g. ClC-2, phospholemman and the channel in Xenopus oocytes.

Animals↗

PubMedDate not supplied

Identification of ClC-2-like chloride currents in pig pancreatic acinar cells.

We used the whole-cell patch-clamp technique to identify a hyperpolarization-activated Cl- current (approximately 50 pA/pF at -60 mV) in acutely isolated, voltage-clamped, single, pig pancreatic acinar cells. This current had characteristic properties of inward rectification, a Cl- = Br->I- selectivity sequence and activation by extracellular hypotonicity. These properties are similar to those reported for the ClC-2 Cl- channel recently cloned from rat and expressed in oocytes. An antiserum raised against the C-terminus of ClC-2 localized the channel to secretory granules containing amylase that were situated exclusively at the apical pole of the pig pancreatic acinar cells, but the channel was not localized in the basolateral membrane. Our study combines a functional assessment and immunohistochemical localization of ClC-2-like channels in a native mammalian cell. The data suggest that the ClC-2-like Cl- channel may function as a Cl- efflux pathway in pancreatic acinar cells.

Animals↗

PubMed1998

Comparison of voltage-activated Cl- channels in rat parotid acinar cells with ClC-2 in a mammalian expression system.

Rat parotid acinar cells express Cl- currents that are activated in a time-dependent manner by hyperpolarized potentials. ClC-2, a member of the ClC gene family, codes for a voltage-gated, inward rectifying anion channel when expressed in Xenopus oocytes. In the present study, we found that cDNA derived from individual parotid acinar cells contained sequence identical to that reported for ClC-2 in rat brain and heart. A polyclonal antibody generated against the N-terminal cytoplasmic domain of ClC-2 recognized an approximately 100 kD protein on western blots of both brain and parotid gland. ClC-2 expressed in oocytes has different kinetics from the currents found in parotid acinar cells. Since the ClC-2 channel was cloned from and its transcripts are expressed in mammalian tissue, we compared the channel properties of acinar cells to a mammalian expression system. We expressed ClC-2 channels in human embryonic kidney cells, HEK 293, using recombinant ClC-2 DNA and ClC-2 DNA fused with DNA coding for jellyfish green fluorescent protein (GFP). Confocal microscopy revealed that the expressed ClC-2-GFP chimera protein localized to the plasma membrane. Whole cell Cl- currents from HEK 293 cells expressing ClC-2-GFP were similar, if not identical, to the Cl- currents recorded from cells transfected with ClC-2 cDNA (no GFP). The voltage-dependence and kinetics of ClC-2 channels expressed in HEK 293 cells were quite similar to those in acinar cells. Channels in parotid acinar and HEK 293 cells activated at more positive membrane potentials and with a faster time course than the channels expressed in Xenopus oocytes. In summary, we found that ClC-2 message and protein are expressed in salivary cells and that the properties of voltage-activated, inward rectifying Cl- channels in acinar cells are similar to those generated by the ClC-2-GFP construct expressed in HEK 293 cells. The properties of the ClC-2 anion channel seem to be dependent on the type of cell background in which it is expressed.

Animals↗

PubMed1994

Characterization of the Cl- conductance in the granular duct cells of mouse mandibular glands.

We have previously shown that mouse mandibular granular ducts contain a hyperpolarization-activated Cl- conductance. We now show that the instantaneous current/voltage (I/V) relation of this Cl- conductance is inwardly rectifying with a slope conductance of 15.4 +/- 1.8 nS (n = 4) at negative potentials and of 6.7 +/- 0.9 nS (n = 4) at positive potentials. Thus, the inward rectification seen in the steady-state I/V relation is due, not only to voltage activation of the Cl- conductance, but also to the intrinsic conductance properties of the channel. We show further that the ductal Cl- conductance is not activated by including ATP (10 mmol/l) in the pipette solution. Finally, we show that the conductance is not blocked by the addition of any of the following compounds to the extracellular solution: anthracene-9-carboxylate (A9C, 1 mmol/l), diphenylamine-2-carboxylate (DPC, 1 mmol/l), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 mumol/l), 4,4'-diisothiocyanato-stilbene-2,2'-disulphonate (DIDS, 100 mumol/l), indanyloxyacetic acid (IAA-94, 100 mumol/l), verapamil (100 mumol/l), glibenclamide (100 mumol/l) and Ba2+ (5 mmol/l). The properties of the ductal Cl- conductance most nearly resemble those of the ClC-2 channel. Both channel types have instantaneous I/V relations that are slightly inwardly rectifying, are activated by hyperpolarization with a time-course in the order of hundreds of milliseconds, have a selectivity sequence of Br- > Cl- > I-, and are insensitive to DIDS. The only identified difference between the two is that the ClC-2 channel is 50% blocked both by DPC and A9C (1 mmol/l), whereas the ductal Cl- conductance is insensitive to these compounds.

Animals↗

PubMed1996

Three distinct chloride channels control anion movements in rat parotid acinar cells.

1. We used the whole-cell configuration of the patch clamp technique to examine the different macroscopic Cl- currents present in single rat parotid acinar cells. 2. Cell swelling produced by negative osmotic pressure (hypotonic bath solutions) induced a large outwardly rectifying Cl- current with little or no time and voltage dependence. In contrast, an increase in intracellular [Ca2+] induced by ionomycin activated Cl- currents with very different properties. Ca(2+)-activated Cl- currents showed outward rectification, relatively slow activation kinetics and marked voltage dependence. These results are consistent with the existence of two different outwardly rectifying Cl- channels in rat parotid cells. 3. In conditions designed to eliminate the activation of these two Cl- currents, a third type of current was observed. This third current was activated in a time-dependent manner by hyperpolarized potentials and was about equally permeant to Cl-, I- and Br-. 4. The properties of the hyperpolarization-activated current were similar to those of the cloned ClC-2 channel. Polymerase chain reaction-based methods and ribonuclease protection analyses indicated the presence in parotid gland of mRNA homologous to ClC-2. 5. Individual parotid acinar cells expressed all three types of Cl- channels. Each type of channel may contribute to Cl- efflux in distinct stages of the secretion process depending on the intracellular [Ca2+], cell volume and membrane potential.

Animals↗

PubMed1996

Volume-activated Cl- channels.

1. An increase in cell volume activates, in most mammalian cells, a Cl- current, ICl,vol. This current is involved in a variety of cellular functions, such as the maintenance of a constant cell volume, pH regulation, and control of membrane potential. It might also play a role in the regulation of cell proliferation and in the processes that control transition from proliferation to differentiation. This review focuses on various aspects of this current, including its biophysical characterisation and its functional role for various cell processes. 2. Volume-activated Cl- channels show all outward rectification. Iodide is more permeable than chloride. In some cell types, ICl,vol inactivates at positive potentials. Single channel conductance can be divided mainly into two groups: small (< 5 pS) and medium conductance channels (around 50 pS). 3. The pharmacology and modulation of these channels are reviewed in detail, and suggest the existence of an heterogeneous family of multiple volume-activated Cl- channels. 4. Molecular candidates for this channel (i.e. ClC-2, a member of the ClC-family of voltage-dependent Cl- channels, the mdr-1 encoded P-glycoprotein, the nucleotide-sensitive pICln protein and phospholemman) will be discussed.

Animals↗

PubMed1998

Characterization of the hyperpolarization-activated chloride current in dissociated rat sympathetic neurons.

1. Dissociated rat superior cervical ganglion (SCG) neurons have been shown to possess a hyperpolarization-activated inwardly rectifying chloride current. The current was not altered by changes in external potassium concentration, replacing external cations with NMDG (N-methyl-D-glucamine) or by addition of 10 mM caesium or barium ions. 2. The reversal potential of the current was altered by changing external anions. The anion selectivity of the current was Cl- > Br- > I- > cyclamate. All substituted permeant anions also blocked the current. 3. The current was blocked by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), 9AC (anthracene-9-carboxylic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) but was unaffected by SITS (4-acetamido-4'-isothiocyanatostilbene- 2,2'-disulphonic acid) and niflumic acid. The effective blockers were voltage dependent; DIDS and NPPB were more effective at depolarized potentials while 9AC was more effective at hyperpolarized potentials. 4. The current was enhanced by extracellular acidification and reduced by extracellular alkalinization. Reducing external osmolarity was without effect in conventional whole-cell recording but enhanced current amplitude in those perforated-patch recordings where little current was evident in control external solution. 5. The current in SCG neurons was blocked by external cadmium and zinc. ClC-2 chloride currents expressed in Xenopus oocytes were also sensitive to block by these divalent ions and by DIDS but the sensitivity of ClC-2 to block by cadmium ions was lower than that of the current in SCG neurons. 6. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed the presence of mRNA for ClC-2 in SCG neurons but not in rat cerebellar granule cells which do not possess a hyperpolarization-activated Cl- current. 7. The data suggest that ClC-2 may be functionally expressed in rat SCG neurons. This current may play a role in regulating the internal chloride concentration in these neurons and hence their response to activation of GABAA receptors.

Animals↗

PubMed1996

Characterization of volume-activated chloride currents in endothelial cells from bovine pulmonary artery.

We have measured the kinetic and pharmacological properties of volume-activated Cl- currents (ICl, vol) in endothelial cells, and tried to correlate them with those of the already described volume-activated current ICln. Both conductances show a similar permeability sequence for monovalent anions, and they are blocked by extracellular ATP. In the present report, we demonstrate by Western blot and RT-PCR that cultured endothelial cells from bovine pulmonary artery (CPAE) contain pICln. The expression of this protein has been shown to be closely associated with the ICln current. ICl, vol showed however, in contrast with ICln, no striking inactivation at positive potentials. This property is also at variance with that of the volume-activated current related to MDR-1. Activation of ICl, vol at potentials more negative than -80 mV was not time dependent, which excludes a major contribution of a ClC-2 related current. The antiviral nucleoside analogue AZT (3'-azido-3'-deoxythymidine) inhibited ICl, vol by 21 +/- 2.7% (n = 10), at a concentration of 100 microM. Another antiviral drug, acyclovir (ACV, 9-[2-hydroxyethoxy) methyl]guanine) blocked ICl, vol by 27 +/- 6.2% at 100 microM (n = 11). Both blocking effects are much smaller than those reported for ICln. The phenol derivative gossypol, which blocks ICln-related currents, efficiently inhibited ICl, vol in CPAE cells (67 +/- 2.1% at 1 microM, n = 7, KI = 0.4 microns). The presence of pICln in CPAE cells and the similar qualitative pharmacological profile of ICl, vol and ICln support the hypothesis that pICln is a good molecular candidate for ICl, vol in endothelial cells. The discrepant kinetic properties may indicate that these time-dependent currents at high positive or negative potentials are not intrinsic properties of the channels, but are caused by time-dependent depletion/accumulation phenomena due to the large amplitudes of these currents.

Animals↗

PubMed1997

Volume expansion-sensing outward-rectifier Cl- channel: fresh start to the molecular identity and volume sensor.

The maintenance of a constant volume in the face of extracellular and intracellular osmotic perturbation is essential for the normal function and survival of animal cells. Osmotically swollen cells restore their volume, exhibiting a regulatory volume decrease by releasing intracellular K+, Cl-, organic solutes, and obligated water. In many cell types, the volume regulatory effluxes of Cl- and some organic osmolytes are known to be induced by swelling-induced activation of anion channels that are characterized by their moderate outward rectification, cytosolic ATP dependency, and intermediate unitary conductance (10-100 pS). Recently, simultaneous measurements of cell size by light microscopy and whole cell Cl- current have shown that the Cl- current density is proportionally increased with an increase in the outer surface area, which is mainly achieved through unfolding of membrane invaginations by volume expansion. Thus this anion channel can somehow sense volume expansion and can be called the volume expansion-sensing outwardly rectifying (VSOR) anion channel. Its molecular identity and activation mechanism are yet to be elucidated. Three cloned proteins, ClC-2, P-glycoprotein, and pIcln, have been proposed as candidates for the VSOR anion channel. The unitary conductance, voltage dependency, anion selectivity, pH dependency, and pharmacology of the VSOR anion channel are distinct from the ClC-2 Cl- channel, which is also known to be sensitive to volume changes. Recent patch-clamp studies in combination with molecular biological techniques have shown that P-glycoprotein is not itself the channel protein but is a regulator of its volume sensitivity. Although there is still debate about another candidate protein, pIcln, the most recent study has suggested that this is likely to be a regulator of some other distinct Cl- channel. Identification of the VSOR anion channel protein per se, its volume-sensing mechanism, and its accessory/regulatory proteins at the molecular level is currently a subject of utmost physiological importance.

Adenosine Triphosphate↗

PubMed1995

PKC-sensitive Cl- channels associated with ciliary epithelial homologue of pICln.

Swelling activates and protein kinase C (PKC) downregulates Cl- channels in cultured nonpigmented ciliary epithelial (NPE) cells. We now report that the PKC inhibitor staurosporine upregulates whole cell Cl- currents isosmotically. The kinetics and current-voltage relationship are similar to those of volume-activated Cl- channels of these cells. These properties are inconsistent with cloned ClC-0, ClC-1, ClC-2, and MDR1 channels but could reflect the cystic fibrosis transmembrane conductance regulator (CFTR) channel or the Cl- channel regulator pICln. CFTR mRNA was undetectable by Northern analysis of cultured NPE cells or ciliary body tissue. In contrast, a human pICln probe obtained by polymerase chain reaction cloning and showing 90% identity with the rat cDNA clone detected high levels of transcripts in NPE cells. The level was low in tissue, where the NPE message was diluted by RNA from other cells. We conclude that NPE cells display staurosporine-activated Cl- channels [gSt(Cl)] likely identical with the volume-activated channels. The same cells expressing gSt(Cl) transcribe mRNA for a novel homologue (pHCBICln) of pICln that may regulate Cl- transport into the aqueous humor.

Alkaloids↗

PubMed1995

Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.

Amino Acid Sequence↗

PubMed1997

Characterization of an inwardly rectifying chloride conductance expressed by cultured rat cortical astrocytes.

The biophysical and pharmacological properties of the inwardly rectifying Cl- conductance (IClh), expressed in rat type-1 neocortical cultured astrocytes upon a long-term treatment (1-3 weeks) with dibutyryl-cyclic-AMP (dBcAMP), were investigated with the whole-cell patch-clamp technique. Using intra- and extra-cellular solutions with symmetrical high Cl- content and with the monovalent cations replaced with N-methyl-D-glucamine, time- and voltage-dependent Cl- currents were elicited in response to hyperpolarizing voltage steps from a holding potential of 0 mV. The inward currents activated slowly and did not display any time-dependent inactivation. The rising phase of the current traces was best fitted with two exponential components whose time constants decreased with larger hyperpolarization. The steady-state activation of IClh was well described by a single Boltzmann function with a half-maximal activation potential at - 62 mV and a slope of 19 mV that yields to an apparent gating charge of 1.3. The anion selectivity sequence was Cl- = Br- = I- > F- > cyclamate > or = gluconate. External application of the putative Cl- channel blockers 4,4 diisothiocyanatostilbene-2,2 disulphonic acid or 4-acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid did not affect IClh. By contrast, anthracene-9-carboxylic acid, as well as Cd2+ and Zn2+, inhibited, albeit with different potencies, the Cl- current. Taken together, these results indicate that dBcAMP-treated cultured rat cortical astrocytes express a Cl- inward rectifier, which exhibits similar but not identical features compared with those of the cloned and heterologously expressed hyperpolarization-activated Cl- channel ClC-2.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Expression of voltage-dependent chloride channels in the rat cochlea.

Voltage-dependent chloride (ClC) channels have not yet been identified in the cochlea. In this study, an approach utilizing the reverse transcription-polymerase chain reaction (RT-PCR) was devised to clone the cDNA of ClC channels. PCR was performed using degenerate primers corresponding to two highly conserved regions of the ClC channels. By Southern hybridization and sequencing studies, the sequences corresponding to ClC-2 and ClC-3 were found in the cochlear lateral wall, while ClC-1 was not detected. These results suggest that ClC-2 and ClC-3 might be involved in Cl- transport in the cochlear lateral wall.

Animals↗

PubMed1996

Cellular and molecular physiology of volume-sensitive anion channels.

Maintenance of a constant cell volume in the face of osmotic stress is an evolutionarily ancient homeostatic process. Over the last two decades physiologists have gained an impressive understanding of the "volume-sensitive" channels, cotransporters, exchangers, metabolic pathways, and genes that are responsible for modulating intracellular solute content and cell volume. This review focuses on one part of this story, the characteristics and osmoregulatory functions of volume-sensitive anion channels. Three distinct types of swelling-activated anion channels have been observed and studied extensively in animal cells. These channels include 1) ClC-2, which is a member of the ClC family of voltage-gated anion channels, 2) an outwardly rectifying intermediate conductance channel, and 3) a large-conductance or "maxi" channel. In addition to these three channels, several other less well-characterized anion channels have been observed. This review discusses the electrophysiological and molecular biological characteristics and regulation of these channels. The possible roles different types of anion channels might play in cell volume homeostasis are also discussed.

Amino Acid Sequence↗

PubMed1997

Psoralens: novel modulators of Cl- secretion.

We evaluated effects of psoralens on Cl- secretion (short-circuit current, I(sc)) across T84 monolayers. Methoxsalen failed to increase I(sc). Several observations suggest that psoralens open cystic fibrosis transmembrane conductance regulator Cl- channels. 1) After activation of the Ca2+-dependent basolateral membrane K+ channel (K(Ca)) by 1-ethyl-2-benzimidazolinone or thapsigargin, methoxsalen (10 microM) further increased I(sc). 2) When added before carbachol (CCh), methoxsalen potentiated the I(sc) response to CCh, as predicted, if it increased apical Cl- conductance. 3) After establishment of a mucosal-to-serosal Cl- gradient and permeabilization of basolateral membrane with nystatin, psoralens increased Cl- current, which was inhibited by glibenclamide. In contrast, neither TS-TM calix[4]arene nor Cd2+, inhibitors of outwardly rectifying Cl- channels and the ClC-2 Cl-channel, respectively, inhibited psoralen-induced Cl- current. In contrast to their effects on Cl- conductance, psoralens failed to significantly affect basolateral membrane K+ conductance; subsequent addition of 1-ethyl-2-benzimidazolinone induced a large increase in K+ conductance. Also, in excised patches, methoxsalen failed to activate K(Ca). In addition to potentiating the peak response to CCh, psoralens induced a secondary, sustained response. Indeed, when added up to 60 min after return of CCh-induced I(sc) to baseline, psoralens induced a sustained I(sc). This sustained response was inhibited by atropine, demonstrating the requirement for continuous muscarinic receptor activation by CCh. This sustained response was inhibited also by verapamil, removal of bath Ca2+, and charybdotoxin. These results suggest that return of I(sc) to baseline after CCh stimulation is not due to downregulation of Ca2+ influx or K(Ca). Finally, we obtained similar results with psoralens in rat colon and primary cultures of murine tracheal epithelium. On the basis of these observations, we conclude that psoralens represent a novel class of Cl- channel openers that can be used to probe mechanisms underlying Ca2+-mediated Cl- secretion.

Animals↗

PubMed1998

Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata.

Membrane hyperpolarization normally activates the slow gate of the Torpedo voltage-gated chloride channel (ClC-0). To elucidate the structural basis of this process, carboxy terminus truncation mutants and chimeras were constructed, expressed in Xenopus oocytes, and evaluated using a two-microelectrode voltage clamp. Introduction of stop codons at several positions between transmembrane domains 12 and 13 (D12 and D13) showed no expression, whereas a truncation just after D13 yielded wild-type currents. A chimera (022) entailing the substitution of the carboxy-terminal cytoplasmic tail after Lys-520 with the corresponding region of ClC-2 lacked slow gating, whereas a more conservative construct (chimera 002), in which D13 was replaced with its ClC-2 analog, retained its capacity to slow gate. These findings suggest that important structures reside within the interdomain stretch (IDS) between D12 and D13. Unlike ClC-2, in which transplantation of "ball" structures could restore gating to constitutively open mutants, transplantation of the ClC-0 IDS to the amino terminus of chimera 022 did not restore gating. Surprisingly, replacement of the IDS by the analogous regions of either ClC-1 or ClC-2 showed slow voltage-activated gating, although the gating was altered. Our findings lead us to conclude that both the functional expression and the slow voltage gating of ClC-0 rely on structures at the carboxy terminus of the channel.

Animals↗