Physiol Rev 82: 503–568, 2002; 10.1152/physrev.00029.2001. Molecular Structure and Physiological Function of Chloride Channels THOMAS J. JENTSCH, VALENTIN STEIN, FRANK WEINREICH, AND ANSELM A. ZDEBIK Zentrum fu¨r Molekulare Neurobiologie Hamburg, Universita¨t Hamburg, Hamburg, Germany I. Introduction 504 II. Cellular Functions of Chloride Channels 506 A. Plasma membrane channels 506 B. Channels of intracellular organelles 507 III. The CLC Chloride Channel Family 508 A. General features of CLC channels 510 B. ClC-0: the Torpedo electric organ ClϪ channel 516 C. ClC-1: a muscle-specific ClϪ channel that stabilizes the membrane voltage 517 D. ClC-2: a broadly expressed channel activated by hyperpolarization, cell swelling, and acidic pH 519 Ϫ E. ClC-K/barttin channels: Cl channels involved in transepithelial transport in the kidney and the Downloaded from inner ear 523 F. ClC-3: an intracellular ClϪ channel that is present in endosomes and synaptic vesicles 525 G. ClC-4: a poorly characterized vesicular channel 527 H. ClC-5: an endosomal channel involved in renal endocytosis 527 I. ClC-6: an intracellular channel of unknown function 531 J. ClC-7: a lysosomal ClϪ channel whose disruption leads to osteopetrosis in mice and humans 531 K. CLC proteins in model organisms 532 on October 3, 2014 IV. Cystic Fibrosis Transmembrane Conductance Regulator: a cAMP-Activated Chloride Channel 533 A. Structure and function of the CFTR ClϪ channel 533 B. Cellular regulation of CFTR activity 534 C. CFTR as a regulator of other ion channels 534 V. Swelling-Activated Chloride Channels 535 A. Biophysical characteristics of swelling-activated ClϪ currents 536 B. Regulation of ICl,swell 536 C. Several molecular candidates for ICl,swell have failed 537 VI. Calcium-Activated Chloride Channels 537 A. Native Ca2ϩ-activated ClϪ channels 537 B. The CLCA (CaCC) family of putative Ca2ϩ-activated ClϪ channels 538 VII. The p64 (CLIC) Gene Family of Putative Intracellular Chloride Channels 539 A. A family of p64-related (CLIC) proteins exists in mammals 540 B. Intracellular distribution and possible functions of CLIC proteins 540 VIII. ␥-Aminobutyric Acid and Glycine Receptors: Ligand-Gated Chloride Channels 541 A. Introduction 541 B. Glycine receptors 543 C. GABAA receptors 544 D. GABAC receptors 546 E. Proteins involved in synaptic localization of GABA and glycine receptors 546 IX. Channel Function in Transporters 547 A. Amino acid transporters 547 B. Phosphate transporters 547 X. Pharmacology of Chloride Channels 548 A. Why bother with pharmacology? 548 B. Mechanisms of ion channel block 548 C. Selective blockers are hard to find: comparison of ClϪ channel classes 549 XI. Outlook 551 Jentsch, Thomas J., Valentin Stein, Frank Weinreich, and Anselm A. Zdebik. Molecular Structure and Physiological Function of Chloride Channels. Physiol Rev 82: 503–568, 2002; 10.1152/physrev.00029.2001.—ClϪ channels reside both in the plasma membrane and in intracellular organelles. Their functions range from ion www.prv.org 0031-9333/02 $15.00 Copyright © 2002 the American Physiological Society 503 504 JENTSCH, STEIN, WEINREICH, AND ZDEBIK homeostasis to cell volume regulation, transepithelial transport, and regulation of electrical excitability. Their physiological roles are impressively illustrated by various inherited diseases and knock-out mouse models. Thus the loss of distinct ClϪ channels leads to an impairment of transepithelial transport in cystic fibrosis and Bartter’s syndrome, to increased muscle excitability in myotonia congenita, to reduced endosomal acidification and impaired endocytosis in Dent’s disease, and to impaired extracellular acidification by osteoclasts and osteopetrosis. The disruption of several ClϪ channels in mice results in blindness. Several classes of ClϪ channels have not yet been identified at the molecular level. Three molecularly distinct ClϪ channel families (CLC, CFTR, and ligand-gated GABA and glycine receptors) are well established. Mutagenesis and functional studies have yielded considerable insights into their structure and function. Recently, the detailed structure of bacterial CLC proteins was determined by X-ray analysis of three-dimensional crystals. Nonetheless, they are less well understood than cation channels and show remarkably different biophysical and structural properties. Other gene families (CLIC or CLCA) were also reported to encode ClϪ channels but are less well characterized. This review focuses on molecularly identified ClϪ channels and their physiological roles. I. INTRODUCTION make comparisons often difficult, this suggests a large molecular diversity of ClϪ channels. ClϪ channels may be Anion channels are proteinaceous pores in biological classified as to their localization (plasma membrane vs. membranes that allow the passive diffusion of negatively vesicular), single-channel conductance, or mechanism of charged ions along their electrochemical gradient. Al- regulation. However, such classification schemes are am- though these channels may conduct other anions (e.g., IϪ biguous. For instance, the same channel may reside in the Ϫ Ϫ Ϫ Downloaded from or NO3 ) better than Cl , they are often called Cl chan- plasma membrane and in intracellular organelles, or the nels because ClϪ is the most abundant anion in organisms mechanisms of activation may overlap. Furthermore, with and hence is the predominant permeating species under the exception of GABA and glycine receptors, such a most circumstances. ClϪ channel gating may depend on classification is unlikely to correlate with the underlying the transmembrane voltage (in voltage-gated channels), gene families. on cell swelling, on the binding of signaling molecules (as The most logical classification of ClϪ channels will in ligand-gated anion channels of postsynaptic mem- be based on their molecular structures. However, the on October 3, 2014 branes), on various ions [e.g., anions, Hϩ (pH), or Ca2ϩ], large variety of biophysically identified ClϪ channels is on the phosphorylation of intracellular residues by vari- not yet matched by a similar number of known ClϪ chan- ous protein kinases, or on the binding or hydrolysis nel genes, suggesting that entire gene families of anion of ATP. channels remain to be discovered. For instance, we prob- Like other ion channels, ClϪ channels may perform ably do not yet know the gene encoding the channel Ϫ their functions in the plasma membrane or in membranes mediating the swelling-activated Cl current (ICl,swell) of intracellular organelles. On the one hand, these func- (volume-sensitive organic anion channel, volume-regu- tions are related to the transport of charge, i.e., to the lated anion channel), and many investigators would agree electric current flowing through the channel, and on the that the genes encoding the archetypal Ca2ϩ-activated ClϪ other hand to the transport of matter. For instance, channels have not yet been identified. plasma membrane ClϪ currents are important for the The correlation of a cloned gene with an ion channel regulation of excitability in nerve and muscle. Currents function is often problematic due to the presence of en- flowing through intracellular ClϪ channels are thought to dogenous channels in the expression system. For in- ensure the overall electroneutral transport of the electro- stance, it now appears that neither mdr (652) nor pICln genic Hϩ-ATPase that acidifies several intracellular com- (469) represents the swelling-activated ClϪ channel (460, partments. On the other hand, bulk flow of chloride is 490). Furthermore, several reports on currents elicited by important for cell volume regulation, as well as for trans- CLC proteins (which form a well-established ClϪ channel epithelial transport. Unlike Ca2ϩ,ClϪ does not seem to family) have probably described currents that are endog- play a role as intracellular messenger. However, the reg- enous to the expression system (75, 127, 359, 366). ulation of ClϪ channel activity by anions (90, 495, 538) So far, we know three well-established gene families also implies that changes in intracellular ClϪ concentra- of ClϪ channels. In mammals, the CLC gene family of Ϫ tion ([Cl ]i) may have a regulatory role. A recent report chloride channels has nine members that may function in (114) additionally suggested that [ClϪ] may serve as an the plasma membrane or in intracellular compartments. allosteric effector in post-Golgi compartments. CLC proteins were thought to have probably 10 or 12 Patch-clamp studies have revealed a bewildering va- transmembrane domains (Fig. 1A, top). This model has riety of anion channels that differ in their single-channel now to be revised because Dutzler et al. (131a) recently conductance, anion selectivity, and mechanism of regula- reported the three-dimensional crystal structure of bacte- tion. Although differences in experimental conditions rial CLC proteins (Fig. 1A, bottom). As already indicated Physiol Rev • VOL 82 • APRIL 2002 • www.prv.org CHLORIDE CHANNELS 505 FIG. 1. Topology models for the es- tablished ClϪ channel families. A, top: CLC ClϪ channel model based on bio- chemical analysis (552). Conflicting re- sults exist in the D4/D5 region (156). The broad hydrophobic region between D9 and D12 was difficult to investigate ex- perimentally, but it was clear that it has an odd number of membrane crossings. The carboxy terminus of all eukaryotic CLC proteins has two CBS domains (30, 484) that have a so far unspecified role in protein-protein interaction. ClC-K pro- teins associate with the ␤-subunit bart- tin, which spans the membrane twice (147) (shown at right). A, bottom: model of CLC ClϪ channel derived from three- dimensional crystal structure of a bacte- Downloaded from rial CLC protein shows that the mem- brane-associated part of the protein is composed of 17 ␣-helices (helix A is not inserted into the membrane). Inspection of the crystal (131a) reveals that most of these helices are not perpendicular to the membrane, but severely tilted. Many of these helices do not span the width of on October 3, 2014 the bilayer. This even serves an impor- tant function, as ClϪ is coordinated in the pore by helices extending from either side of the membrane into the center plane.