Alessandra Picollo

Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel

Phospholipid scramblases disrupt the lipid asymmetry of the plasma membrane, externalizing phosphatidylserine to trigger blood coagulation and mark apoptotic cells. Recently, members of the TMEM16 family of Ca2+-gated channels have been shown to be involved in Ca2+-dependent scrambling. It is however controversial whether they are scramblases or channels regulating scrambling. Here we show that purified afTMEM16, from Aspergillus fumigatus, is a dual-function protein: it is a Ca2+-gated channel, with characteristics of other TMEM16 homologues, and a Ca2+-dependent scramblase, with the expected properties of mammalian phospholipid scramblases. Remarkably, we find that a single Ca2+ site regulates separate transmembrane pathways for ions and lipids. Two other purified TMEM16-channel homologues do not mediate scrambling, suggesting that the family diverged into channels and channel/scramblases. We propose that the spatial separation of the ion and lipid pathways underlies the evolutionary divergence of the TMEM16 family, and that other homologues, such as TMEM16F, might also be dual-function channel/scramblases.

Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters

Ion binding to secondary active transporters triggers a cascade of conformational rearrangements resulting in substrate translocation across cellular membranes. Despite the fundamental role of this step, direct measurements of binding to transporters are rare. We investigated ion binding and selectivity in CLC-ec1, a H+-Cl− exchanger of the CLC family of channels and transporters. Cl− affinity depends on the conformation of the protein: it is highest with the extracellular gate removed and weakens as the transporter adopts the occluded configuration and with the intracellular gate removed. The central ion-binding site determines selectivity in CLC transporters and channels. A serine-to-proline substitution at this site confers NO3− selectivity upon the Cl−-specific CLC-ec1 transporter and CLC-0 channel. We propose that CLC-ec1 operates through an affinity-switch mechanism and that the bases of substrate specificity are conserved in the CLC channels and transporters.

TMEM16 proteins: unknown structure and confusing functions

The TMEM16 family of membrane proteins, also known as anoctamins, plays key roles in a variety of physiological functions that range from ion transport to phospholipid scrambling and to regulating other ion channels. The first two family members to be functionally characterized, TMEM16A (ANO1) and TMEM16B (ANO2), form Ca(2+)-activated Cl(-) channels and are important for transepithelial ion transport, olfaction, phototransduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. The roles of other family members, such as TMEM16C (ANO3), TMEM16D (ANO4), TMEM16F (ANO6), TMEM16G (ANO7) and TMEM16J (ANO9), remain poorly understood and controversial. These homologues were reported to be phospholipid scramblases, ion channels, to have both functions or to be regulatory subunits of other channels. Mutations in TMEM16F cause Scott syndrome, a bleeding disorder caused by impaired Ca(2+)-dependent externalization of phosphatidylserine in activated platelets, suggesting that this homologue might be a scramblase. However, overexpression of TMEM16F has also been associated with a remarkable number of different ion channel types, raising the possibility that this protein might be involved in both ion and lipid transports. The recent identification of an ancestral TMEM16 homologue with intrinsic channel and scramblase activities supports this hypothesis. Thus, the TMEM16 family might have diverged in two or three different subclasses, channels, scramblases and dual-function channel/scramblases. The structural bases and functional implication of such a functional diversity within a single protein family remain to be elucidated and the links between TMEM16 functions and human physiology and pathologies need to be investigated.

Purified TMEM16A is sufficient to form Ca2+-activated Cl− channels

Significance Calcium-activated chloride channels play key roles in physiology, from mediating sensory transduction and nociception to regulating mucine secretion in airway epithelia and controlling excitability of smooth muscle fibers. Recently, TMEM16A was identified as the pore-forming subunit of these channels. It remains unclear, however, whether this protein is sufficient to form calcium-activated chloride channels, or whether association with other subunits is required for function. Recently, association with calmodulin has been proposed to be required for the calcium-dependent activation and ion selectivity of these channels. Here we show that purified and reconstituted TMEM16A is necessary and sufficient to recapitulate the properties of native and heterologously expressed calcium-activated chloride currents. Thus, association of TMEM16A with other proteins is not required for function. Ca2+-activated Cl− channels (CaCCs) are key regulators of numerous physiological functions, ranging from electrolyte secretion in airway epithelia to cellular excitability in sensory neurons and muscle fibers. Recently, TMEM16A (ANO1) and -B were shown to be critical components of CaCCs. It is still unknown whether they are also sufficient to form functional CaCCs, or whether association with other subunits is required. Recent reports suggest that the Ca2+ sensitivity of TMEM16A is mediated by its association with calmodulin, suggesting that functional CaCCs are heteromultimers. To test whether TMEM16A is necessary and sufficient to form functional CaCCs, we expressed, purified, and reconstituted human TMEM16A. The purified protein mediates Ca2+-dependent Cl− transport with submicromolar sensitivity to Ca2+, consistent with what is seen in patch–clamp experiments. The channel is synergistically gated by Ca2+ and voltage, so that opening is promoted by depolarizing potentials. Mutating two conserved glutamates in the TM6-7 intracellular loop selectively abolishes the Ca2+ dependence of reconstituted TMEM16A, in a manner similar to what was reported for the heterologously expressed channel. Well-characterized CaCC blockers inhibit Cl− transport with Kis comparable to those measured for native and heterologously expressed CaCCs. Finally, direct physical interactions between calmodulin and TMEM16A could not be detected in copurification experiments or in functional assays. Our results demonstrate that purified TMEM16A is necessary and sufficient to recapitulate the biophysical and pharmacological properties of native and heterologously expressed CaCCs. Our results also show that association of TMEM16A with other proteins, such as calmodulin, is not required for function.

CLC channels and transporters: proteins with borderline personalities.

Controlled chloride movement across membranes is essential for a variety of physiological processes ranging from salt homeostasis in the kidneys to acidification of cellular compartments. The CLC family is formed by two, not so distinct, sub-classes of membrane transport proteins: Cl(-) channels and H(+)/Cl(-) exchangers. All CLCs are homodimers with each monomer forming an individual Cl- permeation pathway which appears to be largely unaltered in the two CLC sub-classes. Key residues for ion binding and selectivity are also highly conserved. Most CLCs have large cytosolic carboxy-terminal domains containing two cystathionine beta-synthetase (CBS) domains. The C-termini are critical regulators of protein trafficking and directly modulate Cl- by binding intracellular ATP, H+ or oxidizing compounds. This review focuses on the recent mechanistic insights on the how the structural similarities between CLC channels and transporters translate in unexpected mechanistic analogies between these two sub-classes.

Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H + /Cl − exchanger

Active exchangers dissipate the gradient of one substrate to accumulate nutrients, export xenobiotics and maintain cellular homeostasis. Mechanistic studies have suggested that two fundamental properties are shared by all exchangers: substrate binding is antagonistic, and coupling is maintained by preventing shuttling of the empty transporter. The CLC H+/Cl− exchangers control the homeostasis of cellular compartments in most living organisms, but their transport mechanism remains unclear. We show that substrate binding to CLC-ec1 is synergistic rather than antagonistic: chloride binding induces protonation of a crucial glutamate. The simultaneous binding of H+ and Cl− gives rise to a fully loaded state that is incompatible with conventional transport mechanisms. Mutations in the Cl− transport pathway identically alter the stoichiometries of H+/Cl− exchange and binding. We propose that the thermodynamics of synergistic substrate binding, rather than the kinetics of conformational changes and ion binding, determine the stoichiometry of transport.

A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels

The two human CLC Cl− channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca2+ and H+ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca2+]ext without full saturation up to 50 mM. However, in the absence of Ca2+, ClC-Ka currents were still 20% of currents in 10 mM [Ca2+]ext, demonstrating that Ca2+ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H+]ext with a practically complete block at pH 6. Ca2+ and H+ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca2+ assuming a 13-fold higher Ca2+ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca2+ and H+. A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca2+-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca2+. Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H+ block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H+ -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing conformational changes underlying the complex gating of CLC-K channels.

Conformational changes required for H+/Cl− exchange mediated by a CLC transporter

CLC-type exchangers mediate transmembrane Cl− transport. Mutations altering their gating properties cause numerous genetic disorders. However, their transport mechanism remains poorly understood. In conventional models, two gates alternatively expose substrates to the intra- or extracellular solutions. A glutamate was identified as the only gate in the CLCs, suggesting that CLCs function by a nonconventional mechanism. Here we show that transport in CLC-ec1, a prokaryotic homolog, is inhibited by cross-links constraining movement of helix O far from the transport pathway. Cross-linked CLC-ec1 adopts a wild-type–like structure, indicating stabilization of a native conformation. Movements of helix O are transduced to the ion pathway via a direct contact between its C terminus and a tyrosine that is a constitutive element of the second gate of CLC transporters. Therefore, the CLC exchangers have two gates that are coupled through conformational rearrangements outside the ion pathway.

Proton block of the CLC-5 Cl-/H+ exchanger.

CLC-5 is a H+/Cl− exchanger that is expressed primarily in endosomes but can traffic to the plasma membrane in overexpression systems. Mutations altering the expression or function of CLC-5 lead to Dent’s disease. Currents mediated by this transporter show extreme outward rectification and are inhibited by acidic extracellular pH. The mechanistic origins of both phenomena are currently not well understood. It has been proposed that rectification arises from the voltage dependence of a H+ transport step, and that inhibition of CLC-5 currents by low extracellular pH is a result of a reduction in the driving force for exchange caused by a pH gradient. We show here that the pH dependence of CLC-5 currents arises from H+ binding to a single site located halfway through the transmembrane electric field and driving the transport cycle in a less permissive direction, rather than a reduction in the driving force. We propose that protons bind to the extracellular gating glutamate E211 in CLC-5. It has been shown that CLC-5 becomes severely uncoupled when SCN− is the main charge carrier: H+ transport is drastically reduced while the rate of anion movement is increased. We found that in these conditions, rectification and pH dependence are unaltered. This implies that H+ translocation is not the main cause of rectification. We propose a simple transport cycle model that qualitatively accounts for these findings.

Conformational Changes Outside the Ion Pathway are required for Transport in a CLC-Type Cl−/H+ Exchanger

The CLC proteins catalyze transport of chloride ions (Cl-) through cellular membranes in muscle, kidney, bone, and neurons. While some CLCs are ion channels others are H+-coupled secondary active transporters mediating the stoichiometric exchange of 2 Cl- for 1 H+. The exchange mechanism of the CLCs is unclear. All proposed models postulate that the only conformational changes taking place during transport are the movements of a conserved glutamates side chain in and out of the Cl- permeation pathway. This hypothesis is supported by structural and functional work. However, others have suggested that regions distal to the Cl- pathway might also be involved in transport.To test whether transport entails only local or also global rearrangements we constrained the movement of helices J, O, P and Q, which do not line the Cl- or H+ pathways in CLC-ec1, a CLC prokaryotic homologue. If exchange involves the relative movement of these helices then these constraints should reduce the transport rate. In a cys-less background we introduced pairs of cysteines at different locations in this 4-helix bundle and Hg2+-crosslinked them. All unreacted proteins mediate Cl-/H+ exchange at rates comparable to that of the WT. Reaction with Hg2+ results in a striking pattern: constraining residues facing the extracellular side has no effect, while targeting residues deeper in to the protein induces progressively a more drastic reduction of activity. Finally, constraints placed close to the intracellular side results in virtually inactive transporters. This reduction is not due to a Hg2+-induced distortion of the Cl- pathway: Cl- binding is preserved as is Cl- transport through a cross-linked doubly ungated mutant.Thus we propose that the transport cycle in CLC-ec1 entails the movement of these helices outside of the Cl- transport pathway.