C‐Terminal threonines in the KCC4 K‐Cl cotransporter confer sensitivity to cell volume

Abstract

Cation‐chloride cotransporters exhibit reciprocal responses to cell volume, with activation of K‐Cl cotransporters (KCCs) under hypotonic conditions and of Na‐K‐2Cl cotransporters (NKCC1/2) under hypertonic conditions. N‐Terminal phosphorylation by the WNK‐activated SPAK/OSR1 kinases activates NKCCs; in contrast, the KCCs are inhibited by co‐expressed WNK4/SPAK. To identify the residues responsible for volume sensitivity we have systematically screened KCC4 by mutagenesis of serine/threonine residues, followed by assessment of transport activity in Xenopus oocytes under isotonic and hypotonic conditions, +/− WNK4/SPAK. A compound mutant of all 17 N‐terminal serine and threonines has no activity under isotonic conditions, with preserved swelling‐activated transport and inhibition by WNK4/SPAK. Screening of C‐terminal serines and threonines identified several that confer resistance to WNK4/SPAK; alanine mutants of three C‐terminal threonines are constitutively active under isotonic conditions, with analogous effects in KCC2. However, C‐terminal KCC4 fusion protein is not an in vitro substrate for SPAK fusion protein, which phosphorylates N‐terminal NKCC1 fusion protein. In summary, we have identified three C‐terminal threonines that confer volume and WNK/SPAK sensitivity to KCC4; these residues do not however appear to be direct substrates for the SPAK serine‐threonine kinase.