Tryptophan Scanning Mutagenesis Identifies the Molecular Determinants of Distinct Barttin Functions

Abstract

Background:Barttin is an accessory subunit of renal and inner ear chloride channels. Results: Tryptophan insertion at multiple barttin residues results in non-functional CLC-K/barttin channels with normal intracellular trafficking. Conclusion: Gating of CLC-K/barttin channels is more sensitive to tryptophan insertion in barttin than intracellular trafficking. Significance: Understanding the molecular basis of CLC-K modification by its accessory subunit barttin.

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

“…Cells co-expressing W610X hClC-Kb with barttin exhibited slightly larger mean current amplitudes than cells expressing the channel alone (at Ϫ105 mV: Ϫ0.71 Ϯ 0.10 nA, n ϭ 8; p ϭ 0.02 versus hClC-Kb W610X alone). Previous biochemical and microscopic data have demonstrated that hClC-Kb inserts into the plasma membrane even in the absence of barttin (6,26,28). The absence of measurable currents from cells expressing WT hClC-Kb alone indicates that hClC-Kb remains inactive without barttin (6).…”

“…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). Moreover, scanning mutagenesis demonstrated similar effects of tryptophan insertion at each transmembrane domain of barttin on common gating of rClC-K1 and function of hClC-Ka (28). Taken together, these findings strongly suggest that barttin activates human and rodent CLC-K by the same mechanisms, i.e.…”

“…A significant fraction of WT hClC-Kb channels insert into the plasma membrane of HEK293T and MDCK II cells even in the absence of barttin (6,26,28) (Fig. 4F).…”

“…Barttin appears to bind to ClC-K via multiple hydrophobic interactions between the first transmembrane helix of barttin (28) and helices B and/or J (41). In agreement with this notion W610X does not affect hClC-Kb/barttin function via impaired barttin-hClC-Kb association.…”

“…This essential barttin portion contains the two predicted transmembrane helices (8) that are necessary for association of subunit and channel (28,60), the cytosolic amino terminus and 18 amino acids carboxyl-terminal to TM2. Several disease-causing mutations in the cytosolic amino terminus of barttin lack activation of CLC-K channels upon co-expression (26).…”