Design, function, and structure of a monomeric CLC transporter

Channels and transporters of the CLC family bring about transmembrane movement of inorganic anions in service of a variety of biological tasks, from the arcane - generating the kilowatt pulses by which electric fish parboil their prey - to the quotidian - acidification of endosomes, vacuoles, and lysosomes1. The homodimeric architecture of CLC proteins (Fig 1), initially inferred from single-molecule studies of an elasmobranch Cl(−) channel2 and later confirmed by crystal structures of bacterial Cl(−)/H(+) antiporters3,4, appears to be universal. Moreover, the basic machinery enabling ion movement through these proteins - the aqueous pores for anion diffusion in the channels and the ion-coupling chambers that coordinate Cl(−) and H(+) antiport in the transporters - are contained wholly within each subunit of the homodimer. The near-normal function of a bacterial CLC transporter strait-jacketed by covalent crosslinks across the dimer interface and the behavior of a concatameric human homologue argue that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits5,6. However, this evidence is only inferential, and since examples are known in which quaternary rearrangements of extramembrane CLC domains that contribute to dimerization modulate transport activity7, we cannot declare as definitive a “parallel pathways” picture in which the homodimer consists of two single-subunit transporters operating independently. A strong prediction of such a view is that it should in principle be possible to obtain a monomeric CLC. In this study, we exploit the known structure of a CLC Cl(−)/H(+) exchanger, CLC-ec1 from E. coli, to design mutants that destabilize the dimer interface while preserving both structure and transport function of individual subunits. The results demonstrate that the CLC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not required for Cl(−)/H(+) exchange in CLC transporters.