A mutation linked with Bartter's syndrome locks Kir 1.1a (ROMK1) - Channels in a closed state

Mutations in the inward rectifying renal K+ channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. O ne disease-causing mutation removes the last 60 amino acids (332-391), impl icating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated ch annels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles o f this domain were systematically evaluated. When coexpressed with wild-typ e subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion constr uct tvas indistinguishable from the GFP-wild-type channel, demonstrating th at mutant channels are expressed on the oocyte plasma membrane in a noncond uctive or locked-closed conformation. Incremental reconstruction of the COO H terminus identified amino acids 332-351 as the critical residues for rest oring channel activity and uncovered the nature of the functional defect. M utant channels that are truncated at the extreme boundary of the required d omain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of th e Kir 1.1a 331X dominant negative effect suggests a molecular mechanism und erlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant n egative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies t he extreme COOH terminus as an important subunit interaction domain, contro lling the efficiency of oligomerization. Collectively, these observations p rovide a mechanistic basis for the loss of function in one particular Bartt er's-causing mutation and identify a structural element that controls open- state occupancy and determines subunit oligomerization. Based on the overla pping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.