Functional Characterization of Mutations in Kir4.1 (KCNJ10) Associated with the SeSAME Syndrome

Abstract

Kir4.1 channels are expressed in many brain cells, particularly astrocytes, and may be responsible for the K+ buffering action of the glia (J Biol Chem 270: 16339-46, 1995). In addition, Kir4.1 channels are found in the basolateral membrane of distal convoluted tubule cells, where they contribute to renal electrolyte homeostasis. Mutations in KCNJ10, the gene encoding Kir4.1, have been associated to the newly described SeSAME syndrome (Proc Natl Acad Sci USA 106: 5842-47, 2009), a unique set of symptoms that include sensorineural deafness, ataxia, mental retardation and electrolyte imbalance. To determine the functional significance of these mutations, we performed radiotracer efflux assays and inside-out membrane patch clamping in COSm6 cells expressing wild-type (WT) or mutant (R65P, C140R, T164I, A167V, R199Stop, and R297C) channels. All mutations lead to varying degrees of loss of Kir4.1 channel function. In untransfected cells, the 86Rb efflux rate constant was 0.008 min−1 ± 0.001 (n=3), and in cells transfected with WT, the rate of Kir4.1-mediated 86Rb efflux (proportional to K+ conductance) was 0.018 min−1 ± 0.001 (n=3). The mutant Kir4.1-mediated rate constants were 60% (A167V), 21% (R297C), 20% (R65P), 15% (C140R), 12% (T164I), and 1% (R199Stop), relative to WT. No measurable currents were recorded from cells expressing C140R, T164I, R199Stop or R297C. Some of these mutations (R297C, R199Stop) are away from the channel pore, and ongoing studies are examining the potential for altered trafficking. In R65P and A167V, on-cell inward rectification, as well as sensitivity to block by spermine and barium were normal. However, while the current amplitude was similar to WT immediately upon patch excision, it decreased 50-80% within the first 2 min, suggesting that these mutations, located in the potential PIP2 binding site or at the PIP2-dependent gate, reduce open state stability.