Barttin Activates ClC-K Channel Function by Modulating Gating

Abstract

Barttin is an accessory subunit that modifies protein stability, subcellular distribution, and voltagedependent gating of ClC-K chloride channels expressed in renal and inner ear epithelia. ClC-K channels are double-barreled channels with two identical protopores that may be opened by individual or common gating processes. Using heterologous expression in mammalian cells and patch-clamp recordings, we studied the effects of barttin on gating of rat ClC-K1 and human ClC-Ka. In the absence of barttin, rClC-K1 channels displayed two gating processes with distinct kinetics and voltage dependence. A fast gating process, activated by membrane hyperpolarization, opens and closes individual rClC-K1 protopores. In addition, slow common gating steps, stimulated by membrane depolarization, act on both protopores together. Coexpression of barttin results in voltage-independent open probabilities of the common gate, causing increased channel activity at physiologic potentials. In contrast to rClC-K1, human ClC-Ka is functional only when coexpressed with barttin. Single-channel recordings of hClC-Ka/barttin show double-barreled channels with fast protopore gating without apparent cooperative gating steps. These findings demonstrate that barttin stimulates chloride flux through ClC-K channels by modifying cooperative gating of the double-barreled channels and highlight a physiologic role for gating of epithelial ClC chloride channels.

Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking

“…The rodent CLC-K isoform rClC-K1 is active also in the absence of barttin and exhibits fast protopore and slow common gating steps. Co-expression of rClC-K1 and barttin results in a permanently open common gate (27,45). Although such effect on the common gate of human CLC-K channel function cannot be directly demonstrated, we recently demonstrated that hClC-Ka/barttin channels exhibit an open common gate (27).…”

“…This mutation endows rClC-K1 with pronounced barttin-dependent changes in time-and voltage dependence (Fig. 5, A-C) (6,27).…”

“…There are two structurally distinct gating processes, protopore gating that opens and closes individual protopores independently of the adjacent subunit and common gating jointly acting on both pores at once. For such channels, the mean whole-cell current amplitude (I) and the variance 2 depend on the number of channels N, the single channel amplitude of an individual protopore i, as well as on the open probabilities of the protopore and the common gate (P p and P c ) (1,27,38):…”

“…However, in contrast to hClC-Ka and hClC-Kb, rClC-K1 can form functional channels also in the absence of barttin (4,6,7). This property permits studying the effects of barttin on rClC-K1 gating and allowed identification of mechanisms underlying the functional CLC-K modification by barttin (27,45). We decided to use rClC-K1 carrying the V166E mutation to characterize the effects of carboxyl-terminal truncations on CLC-K function.…”

“…hClC-Ka and hClC-Kb are non-functional when expressed alone and require barttin to become functional anion channels. This switch is likely mediated by the modification of channel gating (27). The rodent CLC-K isoform rClC-K1 is active also in the absence of barttin and exhibits fast protopore and slow common gating steps.…”