Known structures and unknown mechanisms of TMEM16 scramblases and channels.

The TMEM16 family of membrane proteins is composed of both Ca2+-gated Cl- channels and Ca2+-dependent phospholipid scramblases. The functional diversity of TMEM16s underlies their involvement in numerous signal transduction pathways that connect changes in cytosolic Ca2+ levels to cellular signaling networks. Indeed, defects in the function of several TMEM16s cause a variety of genetic disorders, highlighting their fundamental pathophysiological importance. Here, we review how our mechanistic understanding of TMEM16 function has been shaped by recent functional and structural work. Remarkably, the recent determination of near-atomic-resolution structures of TMEM16 proteins of both functional persuasions has revealed how relatively minimal rearrangements in the substrate translocation pathway are sufficient to precipitate the dramatic functional differences that characterize the family. These structures, when interpreted in the light of extensive functional analysis, point to an unusual mechanism for Ca2+-dependent activation of TMEM16 proteins in which substrate permeation is regulated by a combination of conformational rearrangements and electrostatics. These breakthroughs pave the way to elucidate the mechanistic bases of ion and lipid transport by the TMEM16 proteins and unravel the molecular links between these transport activities and their function in human pathophysiology.

Out-of-the-groove transport of lipids by TMEM16 and GPCR scramblases.

Phospholipid scramblases externalize phosphatidylserine to facilitate numerous physiological processes. Several members of the structurally unrelated TMEM16 and G protein-coupled receptor (GPCR) protein families mediate phospholipid scrambling. The structure of a TMEM16 scramblase shows a membrane-exposed hydrophilic cavity, suggesting that scrambling occurs via the ?credit-card" mechanism where lipid headgroups permeate through the cavity while their tails remain associated with the membrane core. Here we show that afTMEM16 and opsin, representatives of the TMEM16 and GCPR scramblase families, transport phospholipids with polyethylene glycol headgroups whose globular dimensions are much larger than the width of the cavity. This suggests that transport of these large headgroups occurs outside rather than within the cavity. These large lipids are scrambled at rates comparable to those of normal phospholipids and their presence in the reconstituted vesicles promotes scrambling of normal phospholipids. This suggests that both large and small phospholipids can move outside the cavity. We propose that the conformational rearrangements underlying TMEM16- and GPCR-mediated credit-card scrambling locally deform the membrane to allow transbilayer lipid translocation outside the cavity and that both mechanisms underlie transport of normal phospholipids.

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.

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

Phospholipid (PL) 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 Ca(2+)-gated channels have been shown to be involved in Ca(2+)-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 Ca(2+)-gated channel, with characteristics of other TMEM16 homologues, and a Ca(2+)-dependent scramblase, with the expected properties of mammalian PL scramblases. Remarkably, we find that a single Ca(2+) 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.

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.

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 NO(3)(-) 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.