Large movement in the C terminus of CLC-0 chloride channel during slow gating

Abstract

Chloride channels and transporters of the CLC gene family are expressed in virtually all cell types and are crucial in the regulation of membrane potential, chloride homeostasis and intravesicular pH. There are two gating processes that open CLC channels-fast and slow. The fast gating process in CLC channels has recently been linked to a small movement of a glutamate side chain. However, the molecular mechanism underlying the slow gating process is still elusive. Using spectroscopic microscopy, we observed a large backbone movement in the C terminus of the CLC-0 chloride channel that was functionally linked to slow gating. We further showed that the C-terminal movement had a time course similar to slow gating. In addition, a mutation known to lock the slow gate open prevented movement of the C terminus. When combined with recent structural information on the CLC C terminus, our findings provide a structural model for understanding the conformational changes linked to slow gating in CLC transport proteins.

Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1

“…Hemoglobin is the most famous example in which an oligomeric structure underlies cooperativity (21), allowing a steep relationship between oxygen saturation and oxygen partial pressure. In ClC Cl Ϫ channels one form of gating might arise from its dimeric architecture (22). Thus, oligomeric architectures in both soluble and membrane proteins can allow for much more than the multiplicity of active sites.…”

Molecular determinants of common gating of a ClC chloride channel

“…Common gating of ClC-1 channels is medically important because the vast proportion of ClC-1 mutations that cause myotonia congenita affect the voltage dependence of the common gate 21 . Common gating involves widespread 22,23 cooperative conformational rearrangements that appear to be communicated across the intersubunit interface 24 . Several lines of evidence suggest that, in addition to its obligate role in protopore gating, E ext is also a critical determinant of common gating.…”

CLC Cl − /H + transporters constrained by covalent cross-linking

“…At the other extreme, communication between subunits might be so weak that each functions independently as a fully fledged transporter, a circumstance that would be strikingly analogous to fast gating in the CLC channels. This situation may be argued to apply to the CLC-ec1 transporter, because this protein lacks the C-terminal domain where the common gating movements occur (10). Nevertheless, this fundamental question has not been previously settled and so motivates this work.…”

“…Studies on CLC channels suggest that a large conformational rearrangement of the subunits is directly involved in the common gating process (10,18), but it is not known whether the CLC transporters require similar quaternary movements to move their substrate ions across biological membranes. An extreme possibility of subunit cooperation, for example, might envision an alternating cycle in which one subunit transports Cl Ϫ while the other adopts a different conformation to move H ϩ in the opposite direction.…”

“…In striking contrast, the ''common gating'' process acts cooperatively on both pores, opening or occluding them simultaneously. Because the pores occupy physically distant regions in separate subunits, common gating must involve rearrangement of quaternary structure, i.e., some sort of communication between the subunits; indeed, recent work using fluorescently labeled CLC channels has revealed subunit movements as large as Ϸ20 Å associated with common gating (10).…”