The 57th Annual Meeting and Symposium of the Society of General Physiologists The Biology of Chloride

Olaf S. Andersen

Editor
The Journal of General Physiology


(Organized by H. Criss Hartzell and Michael Pusch)

If for no other reason than the need to maintain electroneutrality, anions are the equals of cations, and chloride channel dysfunctions underlie not only in cystic fibrosis but a host of other diseases. Nevertheless, anions often appear to take the backstage in discussions of membrane transport and ionic homeostasis. Recently, however, they got equal time at the 57th Annual Meeting of the Society of General Physiologists, which took place in Woods Hole, MA, September 3–7, 2003. H. Criss Emory from Emory University of Medicine and Michael Pusch from the CNR Instituto de Biofisica organized the symposium on Biology of Chloride, which highlighted the recent progress that has taken place in understanding the physiological importance of cellular Cl homeostasis and transport. With 196 participants, and 120 invited and poster presentations covering a broad range of topics. Though the topic was profoundly negative, the meeting was very lively.

The meeting's Keynote Speaker, Thomas J. Jentsch (Universität Hamburg, Germany), set the standard with a masterful overview of the mammalian ClC family of chloride channels (or putative chloride channels), which are involved in both plasma membrane and organellar Cl movement (Fig. 1).



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FIGURE 1. The ClC family of mammalian chloride channels. Based on sequence homology, the nine mammalian ClC proteins can be grouped into three classes, of which the first (ClC-1, -ClC-2, ClC-Ka and ClC-Kb) is expressed primarily in plasma membranes, whereas the other two (ClC-3, ClC-4, and ClC-5 and ClC-6 and ClC-7) are expressed primarily in organellar membranes. The primary functions, associated diseases, and phenotype of mouse knockout models are listed where known. After (Jentsch, T.J., V. Stein, F. Weinreich, A.A. Zdebik. 2002. Physiol. Rev. 82:503–568.)

 
The ClC proteins are conserved from bacteria to man and, as is the case for many membrane proteins, atomic resolution structures are known only for the bacterial homologues (Dutzler, R., E.B. Campbell, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 415:287–94.). The membrane-spanning proteins are dimers of subunits with 16 (partial) transmembrane segments; each subunit forms an independent channel (or permeation path). General features of ClC channels are their selectivity for Cl over I (based on conductance measurements) and voltage-dependent gating, which is strongly dependent on the permeant ion concentration. In cases where the gating can be studied in detail, ClC channels have two gates: each subunit is gated by a permeant ion- and voltage-dependent mechanism (the so-called fast gate); in addition, both subunits are gated by a common (or slow) gate.

ClC-1 is the principal skeletal muscle plasma chloride channel, and the classic disease associated with ClC channel dysfunction is Thomsen's disease (myotonia congenita), which results from mutations in the common gate of the mammalian ClC-1 such that the mutation has a strong dominant negative effect. But this is only one of many diseases associated with disrupted Cl transport; among the nine mammalian ClC proteins, five are implicated in human disease (Fig. 1). The physiological role(s) of ClC channels, however, have been difficult to examine because of the lack of specific small-molecule tools to block their function; a limitation that, at least in part, has been overcome by the development of mouse models constructed by gene knockout (Fig. 1).

ClC-2 is a hyperpolarization-activated plasma membrane chloride channel, which is widely expressed but its physiological roles remain enigmatic. A number of inherited epileptic disorders map to the ClC-2 locus, which could suggest that ClC-2 is involved in determining the postsynaptic response to the inhibitory neurotransmitters GABA and Gly. Homozygous ClC-2 knockout mice, however, do not have epilepsy, but suffer from retinal and testicular degeneration, which could suggest that ClC-2 is important for ionic homeostasis in cells (e.g., germ and photoreceptor cells) that depend on close interactions with supporting cells (Sertoli and retinal pigment epithelial cells).

ClC-Ka and ClC-Kb are plasma membrane chloride channels that are expressed predominantly in the kidney and the inner ear; they appear to be unique among the ClC channels in that their assembly and function depends on the ß-subunit barttin. Bartter's syndrome, which also was discussed by F. Hildebrandt (University of Michigan) and S. Uchida (Tokyo Medical and Dental University), is a severe salt-wasting disorder associated with hypokalemic alkalosis, which may be caused by mutations in the apical Na+,K+,2Cl-cotransporter (NKCC2), ROMK, or ClC-Kb. Mutations in barttin, as well as a novel gene product BSND (F. Hildebrandt) causes deafness—in addition to Bartter's syndrome. In the kidney, basolateral ClC-Kb channels are involved in Cl (and K+) reabsorption; in the ear, basolateral ClC-Ka/b channels recycle Cl that enters via the basolateral Na+,K+,2Cl-cotransporter (NKCC1).

The remaining ClC proteins appear to be expressed primarily in intracellular membranes, where they become important for vesicular acidification. Disrupting the function of these proteins therefore will alter many intracellular organelle functions—including endocytosis and intracellular vesicular traffic—and disruptions of organellar ClC protein function may have unexpected consequences. This was illustrated using ClC-5, which was discovered by positional cloning as a candidate gene for Dent's disease—an X-linked disorder associated with low-molecular-weight proteinuria, hypercalciuria, and hyperphosphaturia. How could disrupting the function of a chloride channel cause these changes? ClC-5 colocalizes with the vacuolar proton pump just below the apical plasma membrane in the proximal tubule, and ClC-5 knockout causes proteinuria because it reduces endocytosis (both receptor-mediated and fluid phase) to ~20% of the control level. ClC-5 knockout also disrupts trafficking of the Na+-linked phosphate transporter NaPi-2 by an unexpected mechanism. In most proximal tubule segments the plasma membrane expression of NaPi-2 is reduced rather than increased, as would be expected from a reduced rate of endocytosis. The solution to the puzzle is likely to be that the reduced rate of endocytosis (and associated proteinuria) increases the delivery of parathyroid hormone (PTH) to the distal part of proximal tubule, where PTH binds to apical receptors to activate the endocytosis of NaPi-2 cotransporters. The reduced PTH endocytosis early in the proximal tubule thus may cause reduced phosphate reabsorption late in the proximal tubule—and hyperphosphaturia. The reduced endocytosis of PTH also may account for the hypercalciuria, because the increased delivery of PTH to the late parts of the proximal tubule, with consequent increased binding of PTH to the apical receptors, increases the expression of the a-hydroxylase that converts 25-OH-vitamin D3 to the active 1,25-(OH)2-vitamin D3 to stimulate Ca2+ absorption in the intestine and thereby increase the filtered Ca2+ load to cause hypercalciuria.

The role of ClC-7 was discussed by U. Kornak (Universität Hamburg, Germany). ClC-7 also is a ubiquitous intracellular ClC protein that is expressed especially in bone and the central nervous system. ClC-7 knockout mice are small and die about six weeks after birth. The animals suffer from osteopetrosis due to defective bone resorption resulting from abnormal osteoclast function. In osteoclasts ClC-7 normally is expressed in the ruffled membrane that is formed when H+-ATPase-containing late endosomes fuse with the plasma membranes, whereas ClC-7 is necessary to maintain electroneutrality when H+ is secreted into the resorption lacunae where bone resorption occurs. In the ClC-7 knockout mice, the osteoclasts are present in normal numbers; but when examined by electron microscopy they have poorly developed resorption lacunae, which accounts for the defective bone resorption. The murine ClC-7 knockout phenotype closely resembles the severe infantile malignant osteopetrosis in humans, and ClC-7 mutations are involved in many forms of human osteopetrosis, which indicates that changes in Cl conductance regulate bone resorption—presumably by regulating the acidification rate.

The only ClC proteins for which an atomic resolution structure is known are the bacterial ClC homologues ClC-ec1 and ClC-st1 (from E. coli and S. typhimurium, respectively), which were reviewed by R. Dutzler (The Rockefeller University and Universität Zürich, Switzerland). Crystals were grown in the absence and presence of monoclonal Fab fragments, which increased the resolution with no significant perturbation of the structure. Each ClC subunit has 18 {alpha}-helices, of which 16 are within the bilayer-spanning domain, where their organization differs substantially from that predicted from hydrophobicity analysis (which suggests 12 transmembrane helices). Not only are some helices not predicted by the hydrophobicity analysis, but many helices have unexpected breaks and large tilts relative to the bilayer normal. More detailed analysis shows that the COOH-terminal domain is the antiparallel homologue of the NH2-terminal domain, similar to the organization in the aquaporins. The anion binding sites were identified using Cl -> Br substitution and anomalous scattering. There are two anion binding sites in which the anions are solvated, in part, by the NH2 termini of three {alpha}-helices in a pseudo-threefold symmetric arrangement; but compared with the K+ binding sites in KcsA, the Cl binding sites are fairly hydrophobic. The more extracellular site is close to Glu148, which by sequence alignment corresponds to the functionally important Glu166 in the Torpedo ClC-0. When Glu148 is mutated to Ala or Gln there is a local rearrangement of the structure and a third anion binding site appears at the site of carboxyl side chain, which could suggest that Glu148 may be involved in the anion-dependent channel gating.

A surprising feature of the ClC-ec1 structure is the absence of a well-defined pore for ion movement through the protein. This conundrum was discussed by M. Maduke (Stanford University), who pointed out that any pore would be "hidden" by the cytoplasmic helix R. So, does the pore only "open" after helix R has moved out of the way, to form part of an intracellular access channel? To evaluate this possibility, Lys519 in ClC-0 (which by sequence alignment is in the cytoplasmic helix R) was mutated to Cys, which not only reduced the single-channel conductance but also altered the gating (as would be expected for a permeant ion–dependent gating mechanism). Subsequent modification with the positive-charged Cys reagent methanethiosulfonate ethylammonium tended to normalize channel function, whereas modification with the corresponding negatively charged reagent methanethiosulfonate ethylsulfonate further accentuated the changes in function, suggesting that helix R indeed could line an intracellular access channel.

But, is ClC-ec1 a channel? This question was addressed by C. Miller (Brandeis University), who first pointed out that bacterial ClC preparations tend to be contaminated with porins, which effectively precludes detailed studies of bacterial ClC function. Using highly purified ClC-ec1 preparations, with minimal porin contamination, one can observe discrete current steps when ClC-ec1, reconstituted into phospholipid vesicles, fuse with planar lipid bilayers; but it is impossible to measure discrete single-channel steps after reconstitution into the bilayer (see article by Accardi and Miller, 2004, in this issue). By relating the magnitude of the initial current step to the number of ClC-ec1 dimers, the conductance of each dimer can be estimated to be ~100 fS, corresponding to a unidirectional flux of ~105 ions/s—a number in between the canonical turnover numbers for channels and carriers. The real surprise, however, was that though the current's reversal potential varied as a linear function of log{[Cl]}, the slope was only ~30 mV/decade! Given the purity of the preparation, the inescapable conclusion is that ClC-ec1 catalyzes the transmembrane movement of not only Cl but also some other ion. Which one, and by what mechanism? The "obvious" candidates (K+, OH, and buffer ions) could be ruled out. H+ turned out to be permeant, with a permeability ration PH/PCl {approx} 50, but the current did not show the correct pH dependence. Maybe ClC-ec1 is not a chloride channel after all, but rather an H+/Cl antiport with a 2 H+ for 1 Cl stoichiometry! Though heretical, such a mechanism would provide an explanation for the Cl-dependent gating of the mammalian ClC channels. More generally, the ClC family of proteins may be on the mechanistic interface between carriers and channels—and some mammalian ClC proteins could be carriers (or antiporters) rather than channels.

ClC proteins are important for many cell functions, and many organisms have multiple genes belonging to the ClC family, complicating the mapping of gene product to function. K. Strange (Vanderbilt University) described how C. elegans has six genes, and that an inwardly rectifying chloride channel with properties similar to that of mammalian ClC-2 is expressed in the C. elegans oocytes. The molecular identity of the channel could be determined using RNA-mediated gene interference (RNAi), which in practice was accomplished by feeding the worms RNAi-transfected bacteria. It thus could be shown that the oocyte channel was encoded by clh-3, and that the channel becomes expressed during oocyte meiotic maturation. CLH-3 is localized in the oocyte plasma membrane; it is swelling activated, but plays no role in oocyte volume homeostasis! Oocyte maturation induces ovulatory contractions of electrically coupled gonadal sheath cells. Disrupting CLH-3 function by feeding the worms clh-3 RNAi–transfected bacteria disrupts the timing of the ovulatory contractions, suggesting that the channel modulates ovulation through oocyte-sheath cell intercellular signaling pathways. H. Barbier-Brygoo (Institute des Sciences Végétal, Gif sur Yvette) described how A. thaliana has seven ClC-like genes, and ClC-like proteins are broadly expressed, but none of them appear to function as chloride channels. Their possible functions were explored using gene knockout, and two A. thaliana ClC proteins have been implicated in nitrate metabolism and storage. B. Swappach (Universität Heidelberg, Germany) presented results obtained in yeast, which has only one ClC-related gene that encodes the homologue Gef1p. Gef1p is localized to organelles in the secretory pathway, where it is important for acidification, Ca2+ sequestration, as well as the cellular response to toxic cations and anions. Great care was taken to work at endogenous expression levels, which turned out to be critical because at these low expression levels Gef1P is proteolytically processed, which appears to be important for proper channel trafficking and function. Gef1p forms a complex with a small dimeric ATPase, which is not essential for channel function, but may be important for Gef1p's role in the cellular response to toxic cations.

The importance of protein–protein interactions for chloride channel assembly and targeting was discussed also by H. Betz (Max-Planck-Institute für Hirnforschung), who described how the assembly of the GABAA and glycine receptors is regulated by the submembraneous scaffolding protein gephyrin, which is essential for receptor clustering and normal synaptic activity. K.L. Kirk (University of Alabama) described how the function of CFTR is regulated by a highly conserved amphipathic helix at the NH2-terminal cytoplasmic tail. When this tail is removed, channel activity is reduced to very low levels, but activity can be restored by complementation with the soluble tail. This complementation depends on the presence of F508 in the nucleotide binding domain 1; but even in the {Delta}F508 mutant, overexpression of the tail helps to restore some transport activity, as evaluated by halide flux measurements. It remains unclear whether this partial restoration of activity is due to improved targeting or enhanced function of the few {Delta}F508-CFTR that reaches the plasma membrane.

It has long been known that the survival of CFTR mice depends on the genetic background of the parent strain. As summarized by C. Bear (University of Toronto), the severity of the disease seems to correlate with the level of ClC channel expression—and ClC-2 and ClC-4 are expressed in the apical plasma membrane of the intestine, ClC-2 at the villus apex and ClC-4 in the crypt, suggesting that they could serve as "rescue channels". Definitive evidence is lacking, however, as the available pharmacological tools are insufficient to evaluate their importance for Cl absorption in the CFTR mice—and it remains unclear whether ClC-4 is expressed in the apical plasma membrane.

ClC-3 may well be the most controversial among the ClC proteins. The general agreement is, that ClC-3 is widely expressed in intracellular organelles, and S. Weinman (University of Texas Medical Branch) summarized results on the role of ClC-3 in intravesicular acidification. Several different splice forms occur, with differential localization to different organellar membranes. If ClC-3 is knocked out in murine liver cell lines the endosomal pH is increased, indicating that vesicular acidification indeed is dependent on ClC-3. ClC-3 also may be present in the plasma membrane, but different laboratories have widely differing results. J.R. Hume (University of Nevada School of Medicine) summarized results suggesting that ClC-3 may be involved in cell-volume regulation. The situation is complex because ClC-3 knockout has little effect on volume regulation, whereas ClC-3 antibodies disrupt volume regulation. Though an impressive amount of results were presented, the molecular identity of the channel(s) involved in volume regulation appears to remain an open question.

In comparison to the cation-permeable channels, the pharmacology of chloride channels is lacking. D. Conte-Camerino (University of Bari, Italy) summarized the state of affairs with respect to the pharmacology of ClC channels. Two promising classes of channel inhibitors have been identified, based on 9-anthracenecarboxylic acid (9AC) and 2-(p-chlorophenoxy)propionic acid (CCP) derivatives. CCP is a particularly interesting lead compound, as S(–)CPP blocks ClC-1 in a concentration- and voltage-dependent manner, whereas R(+)CCP has a biphasic effect in which channel activity is enhanced at low concentrations and inhibited at higher concentrations. S(–)CCP is a comparatively selective inhibitor of ClC-1, with a KI {approx} 10 mM and minimal if any effect on ClC-2, ClC-5 and ClC-Ka/b; but anumber of CCP analogs, however, can inhibit the function of various ClC channels, including ClC-Ka, with some specificity. P. Christophersen (Neurosearch, Denmark) summarized results on the development of drugs that could be useful in minimizing the red blood cell (RBC) shrinkage that occurs in sickle cell disease and that may be important in the genesis of the sickle cell crisis. A number of inhibitors of the RBC anion conductance were evaluated; several were found to increase the RBC K+, Na+, and Cl content and reduce the fraction of shrunken cells. Overall, there is reason to be optimistic about the development of new pharmacological tools that can be used for the treatment of human disease as well as for selective modification of chloride channel function.

The biology of Cl spans much further than the ClC family of proteins. D. Oesterhelt (Max-Planck-Institut für Biochemie, Germany) described the passage of Cl through halorhodopsin, a light-driven Cl pump. Halobacteria grow only in very concentrated NaCl solution (>4 M); they have two light-driven ion pumps: bacteriorhodopsin (BR), which is an H+ pump, and halorhodopsin (HR), which can pump Br, Cl, I and nitrate. The physiological need for HR is that halobacteria pumps Cl into the cell in order to increase their volume before cell division, which is an energy-consuming process because of the cytoplasm negative membrane potential. HR is a trimer, in which three palmitates are bound in the center of the trimer where they serve to block noncatalyzed solute movement. Halobacteria and other archea, however, do not synthesize palmitate, which must be taken up from the environment and therefore may be considered to be a vitamin for halobacteria. Four Cl binding sites can be identified in the crystal structure using Br and anomalous scattering; the binding sites are fairly hydrophobic, consistent with the fact that Cl itself is so large that it is somewhat hydrophobic. The photo and transport cycle was elucidated in using mutagenesis and rapid quenching of photo intermediates, which led to the identification of the transport path and the conclusion that the most intracellular Cl in the transport path is released into the cytoplasm before another Cl is taken up at the extracellular entrance to the transport path.

S. Alper (Harvard Medical School) described the role of anion exchangers in cell function. The classic anion exchanger is the RBC Cl/HCO3 exchanger, which is important for the loading/unloading of CO2 in RBCs; but anion exchangers and anion-coupled cation transporters are involved in many other processes, including pH and volume regulation (Fig. 2). Anion exchangers are encoded by the SLC4 and SLC26 gene families, where the SLC4 family includes the Na+-independent, electroneutral exchangers, including the RBC Cl/HCO3 exchanger (AE1), several Na+-HCO3 cotransporters, as well as Na+-dependent anion exchangers. AE1 is important not only for RBC CO2 uptake/release, it also is involved in distal tubular H+ transport, as distal tubular acidosis (dRTA) can result from mutations in the apical H+-ATPase or the basolateral anion exchanger. Surprisingly, however, many AE1 mutations that cause dRTA have only modest effect on RBC anion exchange, which could indicate that the mutations disrupted AE1 trafficking rather than anion exchange per se. In RBCs, AE1 alone can catalyze Cl/Cl exchange; but Cl/HCO3 exchange depends on carbonic anhydrase binding to the cytoplasmic COOH-terminal tail of AE1. B. Fakler (Albert-Ludwigs-Universität Freiburg) summarized recent work on a member of the SLC26 family of anion exchange proteins, prestin, which is involved in regulating the mechanotransduction of hair cells. Some years ago J. Ashmore described an electromechanical response in hair cells, where a change in membrane potential caused a change in hair cell length. The response is fast, up to 50 kHz and ATP and Ca2+ independent, and can be reproduced in systems where SLC26 is expressed heterologously. The response is Cl dependent in the sense that removing the intracellular Cl reduces/abolishes the electromechanical response. A number of organic anions can substitute for Cl, consistent with the SLC26 family of proteins having a broad substrate specificity, and the effect is inhibited by salicylate, an inhibitor of many anion exchangers, but the mechanistic role of the anions in the electromechanical response has not been fully determined.



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FIGURE 2. pH and volume regulatory channels and transporters in nonpolarized cells. Various combinations of channels and transporters can be characterized as being acid and base extruders (in pH regulation) or volume decreasers and increasers (in volume regulation). All four regulatory systems depend on the transmembrane movement of Cl. (Alper, S.L. 1994. Cell. Physiol. Biochem. 4:265–281.)

 
The cation-chloride cotransporters were discussed by B. Forbush (Yale University School of Medicine), who focused on the Na+,K+,2Cl cotransporters NKCC1 and NKCC2, and T. Boettger (Martin-Luther-Universität of Halle-Wittenberg, Germany), who focused on the K+,Cl cotransporters. Both Na+,K+,2Cl cotransporters are involved in the transport of Cl into cytoplasm, and their activity is tightly regulated by phosphorylation, cell volume, and intracellular [Cl] ([Cl]i). Each cotransporter has multiple phosphorylation sites and binding sites for the protein kinase PASK, which appears to be critical for both the volume- and Cl-dependent regulation, as a dominant negative PASK mutant almost completely abolish the ability of NKCC1 and NKCC2 to be up-regulated by decreasing [Cl]i or cell volume. The K+,Cl cotransporters are activated by cell swelling, indicating that they catalyze the efflux of Cl. The neuronal K+,Cl cotransporter KCC2 is essential for regulating [Cl]i in mature neurons by catalyzing the efflux of Cl. Proper KCC2 function therefore is necessary for normal inhibitory synaptic activity. In fact, the increased [Cl]i in KCC2 knock-out mice causes the normally inhibitory neurotransmitters GABA and Gly to excitatory.

A number of ClC proteins are involved in membrane trafficking, but how does the organellar [Cl] actually vary during endocytosis and the subsequent vesicular maturation? A.S. Verkman (University of California, San Francisco) described results obtained with 10,10'-bis[3-carboxypropyl]-9,9'-biacridinium dinitrate, which is a pH-insensitive, ratiometric Cl indicator that can be coupled to various carriers to allow the selective exploration of fluid-phase and receptor-mediated endocytosis. Just after endocytosis the organellar pH is ~7, but [Cl] is only ~20 mM. During the next 30 min or so, the pH decreases and [Cl] increases—and both processes are blocked by vacuolar H+-ATPase inhibitor bafilomycin. These later changes in organellar pH and [Cl] are due to the influx of H+ and Cl in approximate molar equivalence—a coupling that can be abolished by valinomycin—indicating that the organellar membrane's K+ permeability is low. But why is the initial organellar [Cl] so low? Experiments at higher time resolution show that the [Cl] decrease occurs over ~1 min, and that the decrease is diminished by adding polylysine or acidifying the extracellular solution. Therefore, the most likely explanation is that the initial plasma retrieval leads to the formation of tubular structures, in which the fixed negative charges at the intraorganellar membrane–solution interface produce a substantial Gibbs-Donnan effect that excludes Cl. As H+ and Cl move into the cell, water will follow such that the organellar volume increases and the relative importance of the surface charges becomes less, but still remains significant (as the [Cl] remains below that of the extracellular solution).

Methods for measuring Cl were also presented by G.J. Augustine (Duke University Medical Center), who described results obtained using the genetically encoded ratiometric Cl indicator clomeleon, which was developed based on the chance observation that fluorescence of yellow fluorescent protein is quenched by Cl. Transgenic mice were generated in which clomeleon was expressed under the control of the neuronal promoter, to allow for determining changes in [Cl]i during development and normal activity. In pyramidal neurons in slices, [Cl]i {approx} 35 mM at P1 and decreases over the next month to reach ~4 mM at P30, which could be due to maturation of KCC2 function. Activating the GABAA receptors increases [Cl]i and it is possible to monitor the diffusion of Cl within the cells, which occurs more or less as free diffusion. The recovery (Cl extrusion) occurs over 60 s—a time that is long compared with the normal activity rate in hippocampal neurons, meaning that even "resting" [Cl]i will vary as a function of neuronal activity.

The roles of Cl in neuronal function were discussed in several presentations. R.H. Wallace (University of Kentucky Health Science Center) described results on the involvement of the GABAA receptor in epilepsy. Several GABAA mutations cause epilepsy or febrile seizures, many of these mutations cause a reduction in the GABA-induced currents, due to either improper trafficking or reduced receptor function. One mutation, however, located in the benzodiazepine-binding domain, does not alter the current response. Though hardly conclusive, this result could suggest the presence of endozepines. P.R. Schofield (Garvan Institute for Medical Research, Australia) described the role of glycine receptors in human startle disease, which is caused by mutations in the GlyR {alpha}1 subunit. 11 different disease-causing mutations have been described, in which the Gly responses are impaired and the usual agonists ß-alanine and taurine become competitive antagonists or partial agonists. Moreover, a number of disease-causing mutations produce massive shifts in the distribution of subconductance states. Overall, these results suggest that the mutations alter the coupling between agonist binding and channel opening. Using alanine-scanning mutagenesis it could be shown that the allosteric switch that is involved in coupling ligand binding to channel opening involves both extra- and intracellular receptor domains.

A traditional feature of the symposia organized by the Society of General Physiologists is the New Ideas/New Faces sessions, where the speakers are chosen by the organizers based on the free abstracts submitted to the meeting. This is indeed where the new ideas are presented—usually by young investigators. At this meeting J. Arreola (Universidad Autónoma de San Lui Potosí, Mexico) spoke on the nucleotide sensitivity of the ATP-activated chloride conductance in mouse parotid acinar cells, A. Bhattacharjee (Yale University School of Medicine) spoke on slick and slack, which are Cl- and Na+-activated potassium channels found in neurons and other excitable cells, and V. Faundez (Emory University) spoke on adaptor AP-3–dependent targeting of ClC-3 to synaptic vesicles.

Altogether the symposium served to provide a focus for an important set of problems, and the importance of Cl in human health and disease was amply illustrated, which will serve to stimulate a new generation of exciting studies on the biology of Cl.





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