Characterization of the mouse ClC-K1/Barttin chloride channel

Abstract

Several Cl(-) channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl(-) absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl(-)>Br(-)>NO3(-)>I(-). Single-channel recordings revealed a unit conductance of ~40pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~-65mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~20pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~+25mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253-260).

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).…”

“…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.…”

Discovery of CLC transport proteins: cloning, structure, function and pathophysiology

“…Subsequent mutations of the 'gating glutamate' in ClC-0 (Dutzler et al 2003) and CLC exchangers (Leisle et al 2011;Neagoe et al 2010) similarly largely abolished the voltage-dependence of currents. However, channels lacking this glutamate still gate at the single-channel level (Dutzler et al 2003;L'Hoste et al 2013), indicating that CLC gating cannot be explained entirely by the movement of its side chain. Voltage-gating of CLC channels depends on the Cl − concentration.…”

Tryptophan Scanning Mutagenesis Identifies the Molecular Determinants of Distinct Barttin Functions

“…(mClC-K1 and mClC-K2) (4), and humans (hClC-Ka and hClC-Kb) (5) and have been shown to be essential for sensory transduction in the inner ear and for normal urinary concentration. They mediate Cl Ϫ efflux in the thin ascending limb (ClC-K1/ClC-Ka; Refs.…”