Activation of renal ClC-K chloride channels depends on an intact N terminus of their accessory subunit barttin

Abstract

ClC-K channels belong to the CLC family of chloride channels and chloride/proton antiporters. They contribute to sodium chloride reabsorption in Henle's loop of the kidney and to potassium secretion into the endolymph by the stria vascularis of the inner ear. Their accessory subunit barttin stabilizes the ClC-K/barttin complex, promotes its insertion into the surface membrane, and turns the pore-forming subunits into a conductive state. Barttin mutations cause Bartter syndrome type IV, a salt-wasting nephropathy with sensorineural deafness. Here, studying ClC-K/barttin channels heterologously expressed in MDCK-II and HEK293T cells with confocal imaging and patch-clamp recordings, we demonstrate that the eight-amino-acids-long barttin N terminus is required for channel trafficking and activation. Deletion of the complete N terminus (Δ2-8 barttin) retained barttin and human hClC-Ka channels in intracellular compartments. Partial N-terminal deletions did not compromise subcellular hClC-Ka trafficking but drastically reduced current amplitudes. Sequence deletions encompassing Thr-6, Phe-7, or Arg-8 in barttin completely failed to activate hClC-Ka. Analyses of protein expression and whole-cell current noise revealed that inactive channels reside in the plasma membrane. Substituting the deleted N terminus with a polyalanine sequence was insufficient for recovering chloride currents, and single amino acid substitutions highlighted that the correct sequence is required for proper function. Fast and slow gate activation curves obtained from rat V166E rClC-K1/barttin channels indicated that mutant barttin fails to constitutively open the slow gate. Increasing expression of barttin over that of ClC-K partially recovered this insufficiency, indicating that N-terminal modifications of barttin alter both binding affinities and gating properties.

Functional Study of Novel Bartter’s Syndrome Mutations in ClC-Kb and Rescue by the Accessory Subunit Barttin Toward Personalized Medicine

“…According to our electrophysiological, biochemical, and imaging findings, the overexpression of the accessory subunit barttin succeeded in restoring at least in part the function of the expression-defective mutants G167V and A242E towards wild type levels. It is widely demonstrated that barttin is able to improve the stability of ClC-K channels by promoting complex glycosylation of the nascent proteins, thus facilitating effective exit from the ER and Golgi and sorting to the plasma membrane, besides modulating channel gating (Estévez et al, 2001;Waldegger et al, 2002;Hayama et al, 2003;Scholl et al, 2006;Janssen et al, 2009;Fischer et al, 2010;Wojciechowski et al, 2018a;Wojciechowski et al, 2018b). We could therefore likely infer that the same mechanism underlies BS mutant channels rescue.…”

“…Importantly, a molecule able to increase protein stability and prevent ER degradation, such as 17allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, was reported to mitigate the BS symptoms in barttin R8L knock-in mice by likely enhancing the plasma membrane expression of ClC-K1/mutant barttin channels (Nomura et al, 2013). Barttin seems to interact with hydrophobic residues in helices B and J, at the outer surface of the ClC-K channels, through its short N-terminal and first TM1 helix (Tajima et al, 2007;Wojciechowski et al, 2018a).…”

Modelling salt transport disorders of human kidney in zebrafish: the grain of salt

“…; Wojciechowski et al . ). In the human, barttin regulates intracellular trafficking and the activity of both ClC‐Ka and ClC‐Kb.…”

“…The activation of ClC‐Ka and ClC‐Kb is also enhanced by palmitoylation of barttin on its 55 and 56 cysteine residues (Wojciechowski et al . ). Point mutations on these two cysteine residues prevent post‐translational modification of barttin and deplete the currents produced by human ClC‐Ka and ClC‐Kb (Wojciechowski et al .…”

“…Point mutations on these two cysteine residues prevent post‐translational modification of barttin and deplete the currents produced by human ClC‐Ka and ClC‐Kb (Wojciechowski et al . ). Confirming the previous findings, the recent study (Pérez‐Rius et al .…”