Twenty‐five years of CLC chloride transport proteins
Year: 2015
Type: article
Abstract: With this Special Issue, The Journal of Physiology celebrates significant advances enabled in 1990 by molecular identification of the first member of the CLC family of transport proteins – the Torpedo voltage-gated chloride channel, CLC-0. Its peculiar gating properties, first described and modelled by Chris Miller nearly a decade previously, thereby became amenable to further biophysical interrogation. The timing also could not be better, for a year earlier, the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) also was identified. Together, these accomplishments ushered the study of anion transport into the molecular era, thrusting onto centre stage this previously understudied topic. Consequently, through studies utilizing not only biophysics, but also cellular physiology, pharmacology, model systems and genetic analyses, coherent models coalesced to advance understanding of the physiology and pathophysiology of practically every system in the body. Now, a quarter of a century later, it is apropos to take a look back on these discoveries and reflect on what has been learned. It is also a time to survey the horizon and contemplate what lies beyond. In the first review, Chris Miller recounts the deductive processes leading ultimately to the biophysical characterization of the Torpedo voltage-gated chloride channel before molecular identification of the CLC family. At first an unwelcome guest in his bilayer reconstitution experiments of Torpedo californica electroplax membranes, this interloper's enigmatic gating behaviour captured Miller's attention and curiosity. In-depth scrutiny produced his ‘double-barrelled’ model of chloride channel gating: two independently and rapidly gating pores sharing a common ‘slow’ gate (Miller, 1982). This now-classic model established a distinct biophysical signature critical for informing the key functional analyses used later by Jentsch and colleagues (Jentsch et al. 1990). The ‘double-barrelled’ chloride channel model continues to shape the perspective of biophysicists and physiologists currently working on CLC transport proteins. Thomas Jentsch next recollects his laboratory's initial expression cloning of CLC-0 from the electric organ of Torpedo marmorata (the smaller European cousin of T. californica). Importantly, heterologous expression in Xenopus oocytes yielded currents exhibiting the identical curious ‘double-barrelled’ gating behaviour described earlier by Miller. This Herculean achievement launched a decade of prolific discovery. With critical primary structural information in hand, the isolation of cDNAs encoding related proteins became possible, and the subsequent, relentless flurry of activity by the Jentsch lab identified many related CLC family members, starting first with the cloning of CLC-1 through homology screening of a rat skeletal muscle cDNA library. Subsequent mutational linkage analysis and complementary structure–function analyses established that dominant and recessive myotonias in humans and animals result from mutations within the genes encoding CLC-1. As the decade progressed and many additional CLC family members were identified, functional characterizations largely employed similar analytic paradigms. In due time, murine models were developed and implemented, informing (patho)physiological context and mechanism. By the end of the decade (and century), the field had available the initial working models for explaining a constellation of human genetic diseases, including dominant and recessive forms of human myotonia, the renal diseases, Bartter's syndrome and Dent disease, and osteopetrosis. Despite these achievements, many mechanistic details remained unresolved. For example, how might Bartter's syndrome emerge from mutations in the gene (Simon et al. 1997) encoding a questionably functional protein (Uchida et al. 1993; Kieferle et al. 1994)? And how is acidification of intracellular compartments sub-served by the paradoxically outwardly rectifying currents displayed by the intracellular CLCs, notably CLC-5 and CLC-7? What are the structural determinants of gating? These puzzles amply sustained the field's momentum. Indeed, the advent of the 21st century brought a flood of revelations, each shedding light on these issues. Our remaining three Invited Topical Reviews, authored by leading investigators of the next generation, synthesize the impact of these advances in shaping both current and emergent mechanistic models, filling existing gaps as well as providing novel insights. First of these was the discovery that three CLC transport proteins associate specifically with molecularly distinct β subunits that act to regulate function. Barrallo-Gimeno and colleagues review the current state of knowledge regarding these auxiliary subunits: (1) Barttin, important for functionality and regulation of CLC-K channels, (2) GlialCam, which modifies the physiology of its partner, the ubiquitously distributed CLC-2, and (3) Ostm1, critical for CLC-7 activity and stabilization (Barrallo-Gimeno et al. 2015). Identified from analysis of genes linked to Bartter's syndrome (Birkenhäger et al. 2001), Barttin proved essential for functional expression of CLC-Ka and CLC-Kb currents (Estévez et al. 2001). This initial example set by CLC-K/Barttin established an important precedent: CLCs can be regulated by association with auxiliary subunits. Associative interactions include stabilization of CLC-7 with Ostm1, as well as tissue-specific regulation by GlialCAM of CLC-2, a CLC channel which is not strictly dependent on β subunits for function. Barrallo-Gimeno et al thus consider the complex functional relationships between CLCs and their partner subunits, as well as how their associated genetic mutations lead to disease: distinct types of Bartter's syndrome (with and without deafness), leukoencephalopathies, osteopetrosis and neuronal degenerative disorders. Solution of the first high-resolution, three-dimensional crystal structure of a bacterial CLC protein next ushered in a new era of structure–function analysis (Dutzler et al. 2002). Since then, other structures – including one eukaryotic – were obtained. In the fourth review, Alessio Accardi relates how these rich sources of enhanced spatial information have been used to fine-tune structure–function studies, thereby informing detailed mechanistic models of CLC ionic selectivity and transport, as well as gating by intracellular nucleotide binding (Accardi, 2015). The stunning discovery that select CLCs function not as channels, but rather as chloride–proton antiporters, furnished fresh opportunities to re-evaluate their putative physiological function in promoting acidification of intracellular compartments (Accardi & Miller, 2004). How might such an updated function challenge previous models hypothesizing roles for intracellularly localized and (paradoxically) outwardly rectifying CLCs as chloride shunt pathways? Giovanni Zifarelli's contribution to our Special Issue considers the mechanistic implications of alternative models arising from CLC antiporter function (Zifarelli, 2015). He further discusses how liberation of CLCs from the constraints of channel-dom led to recent discovery of additional cellular roles, some of which may involve functions entirely unrelated to ion transport. The last quarter of a century brought landmark advances in understanding the structure, function and regulation of CLC transport proteins, enriching the collective knowledge base of anion transport physiology in both depth and breadth. The influence of these discoveries on related sub-disciplines, as well as on medicine at large, cannot be overestimated. What puzzles remain to be solved and what breakthroughs will the next 25 years bring? Onward! None.
Source: The Journal of Physiology
DOI: 10.1113/jp271325
Author: Peying Fong
Institution: Kansas State University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2159070177