Variable loss of Kir4.1 channel function in SeSAME syndrome mutations

Abstract

SeSAME syndrome is a complex disease characterized by seizures, sensorineural deafness, ataxia, mental retardation and electrolyte imbalance. Mutations in the inwardly rectifying potassium channel Kir4.1 (KCNJ10 gene) have been linked to this condition. Kir4.1 channels are weakly rectifying channels expressed in glia, kidney, cochlea and possibly other tissues. We determined the electrophysiological properties of SeSAME mutant channels after expression in transfected mammalian cells. We found that a majority of mutations (R297C, C140R, R199X, T164I) resulted in complete loss of Kir4.1 channel function while two mutations (R65P and A167V) produced partial loss of function. All mutant channels were rescued upon co-transfection of wild-type Kir4.1 but not Kir5.1 channels. Cell-surface biotinylation assays indicate significant plasma membrane expression of all mutant channels with exception of the non-sense mutant R199X. These results indicate the differential loss of Kir channel function among SeSAME syndrome mutations.

Introduction

The biophysical fingerprint of Kir channels is inward rectification in the current–voltage relationship, which limits K⁺ efflux at depolarizing membrane potentials [1], [2], [3]. By virtue of their functional properties, Kir channels are essential in control of resting membrane potential, coupling of the metabolic cellular state with membrane excitability, and maintenance of K⁺ homeostasis in diverse cell types. Kir4.1 channels encoded by the KCNJ10 gene are moderately rectifying K⁺ channels with expression in the nervous system, inner ear and in the kidney [4], [5], [6], [7], [8], [9]. Previous work has shown that Kir4.1 channel is the principal K⁺ channel expressed in glial cells such as retinal Müller cells, brain astrocytes and satellite glial cells [10], [11], [12], [13], [14], [15]. Moreover, we and others have found that loss of Kir4.1 channel expression in mice leads to profound glial cell membrane depolarization, impaired extracellular K⁺ buffering, and abnormal neuronal activity in the retina and brainstem [15], [16]. The SeSAME (or EAST) syndrome is characterized by seizures, sensorineural deafness, ataxia, mental retardation and electrolyte imbalances and has been mapped to missense or non-sense mutations in the KCNJ10 gene [17], [18]. Functional consequences of SeSAME mutations likely involve the partial or total loss of Kir4.1 channel activity [17], [18]. However, a more comprehensive characterization of the consequences of such mutations for Kir4.1 channel function has yet to be performed. In this study, we report that SeSAME syndrome mutations cause variable loss of Kir4.1 channel function that can be rescued upon co-expression of wild-type Kir4.1 but not with Kir5.1 channels. Furthermore, almost all mutations do not largely impair the ability of these channels to be expressed at the plasma membrane indicating the potential utility of Kir channel openers for SeSAME syndrome therapy.