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 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 PL 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.

Dystroglycan, which serves as a major extracellular matrix receptor in muscle and the central nervous system, requires extensive O-glycosylation to function. We identified a dystroglycan missense mutation (Thr192→Met) in a woman with limb-girdle muscular dystrophy and cognitive impairment. A mouse model harboring this mutation recapitulates the immunohistochemical and neuromuscular abnormalities observed in the patient. In vitro and in vivo studies showed that the mutation impairs the receptor function of dystroglycan in skeletal muscle and brain by inhibiting the post-translational modification, mediated by the glycosyltransferase LARGE, of the phosphorylated O-mannosyl glycans on α-dystroglycan that is required for high-affinity binding to laminin.

CLC-ec1 is a prokaryotic CLC-type Cl⁻/H⁺ exchange transporter. Little is known about the mechanism of H⁺ coupling to Cl⁻. A critical glutamate residue, E148, was previously shown to be required for Cl⁻/H⁺ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H⁺ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H⁺ coupling to Cl⁻ transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl⁻ transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl⁻ transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl⁻ and one for H⁺. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes.

CLC-ec1 is an E. coli homologue of the CLC family of Cl− channels, which are widespread throughout eukaryotic organisms. The structure of this membrane protein is known, and its physiological role has been described, but our knowledge of its functional characteristics is severely limited by the absence of electrophysiological recordings. High-density reconstitution and incorporation of crystallization-quality CLC-ec1 in planar lipid bilayers failed to yield measurable CLC-ec1 currents due to porin contamination. A procedure developed to prepare the protein at a very high level of purity allowed us to measure macroscopic CLC-ec1 currents in lipid bilayers. The current is Cl− selective, and its pH dependence mimics that observed with a 36Cl− flux assay in reconstituted liposomes. The unitary conductance is estimated to be <0.2 pS. Surprisingly, the currents have a subnernstian reversal potential in a KCl gradient, indicating imperfect selectivity for anions over cations. Mutation of a conserved glutamate residue found in the selectivity filter eliminates the pH-dependence of both currents and 36Cl− flux and appears to trap CLC-ec1 in a constitutively active state. These effects correlate well with known characteristics of eukaryotic CLC channels. The E148A mutant displays nearly ideal Cl− selectivity.