Novel KCNJ10 Gene Variations Compromise Function of Inwardly Rectifying Potassium Channel 4.1

Abstract

The KCNJ10 gene encoding Kir4.1 contains numerous SNPs whose molecular effects remain unknown. We investigated the functional consequences of uncharacterized SNPs (Q212R, L166Q, and G83V) on homomeric (Kir4.1) and heteromeric (Kir4.1-Kir5.1) channel function. We compared these with previously characterized EAST/SeSAME mutants (G77R and A167V) in kidney-derived tsA201 cells and in glial cell-derived C6 glioma cells. The membrane potentials of tsA201 cells expressing G77R and G83V were significantly depolarized as compared with WTKir4.1, whereas cells expressing Q212R, L166Q, and A167V were less affected. Furthermore, macroscopic currents from cells expressing WTKir4.1 and Q212R channels did not differ, whereas currents from cells expressing L166Q, G83V, G77R, and A167V were reduced. Unexpectedly, L166Q current responses were rescued when co-expressed with Kir5.1. In addition, we observed notable differences in channel activity between C6 glioma cells and tsA201 cells expressing L166Q and A167V, suggesting that there are underlying differences between cell lines in terms of Kir4.1 protein synthesis, stability, or expression at the surface. Finally, we determined spermine (SPM) sensitivity of these uncharacterized SNPs and found that Q212R-containing channels displayed reduced block by 1 M SPM. At 100 M SPM, the block was equal to or greater than WT, suggesting that the greater driving force of SPM allowed achievement of steady state. In contrast, L166Q-Kir5.1 channels achieved a higher block than WT, suggesting a more stable interaction of SPM in the deep pore cavity. Overall, our data suggest that G83V, L166Q, and Q212R residues play a pivotal role in controlling Kir4.1 channel function.Inwardly rectifying potassium (Kir) 3 channels are involved in maintenance of negative resting membrane potential (1-3), potassium buffering (1, 4), extracellular glutamate clearance (1, 4, 5), myelination (6), and cell volume regulation (3, 7). Kir4.1 channels are tetramers formed by Kir4.1 (KCNJ10) subunits. Kir4.1-containing channels are expressed in glial cells (8 -14) including in astrocytic end feet surrounding blood vessels (9, 15-17) and surrounding neuronal synapses (9, 17) where they are involved in maintenance of extracellular [K ϩ ] o and glutamate homeostasis (4,11,18). These functions are critical as inability to control [K ϩ ] o and glutamate alters neuronal excitability and may lead to seizures and neuronal death (4, 18 -20). Similarly in the retina, Kir4.1-dependent Kir channels are involved in homeostasis of extracellular potassium produced by neuronal activity in a process called potassium siphoning (9, 14, 21, 22).Kir4.1 subunits are also prominently expressed in the distal convoluted tubules in the kidneys (23) where they are involved in K ϩ recycling (24) and in the ear, specifically in the stria vascularis, where they are responsible for producing the endocochlear potential (7). Complete absence or loss-of-function mutations in these channel subunits cause EAST/SeSAME syndrome characterized by seizures, se...

Collecting system–specific deletion of Kcnj10 predisposes for thiazide- and low-potassium diet–induced hypokalemia

“…7). Interestingly, several single nucleotide polymorphisms (SNPs) that change the biophysical properties of Kir4.1 have been described in the KCNJ10 gene 45 . It is tempting to speculate that other unexplained variants of hypokalemia might be linked to polymorphisms in Kcnj10 / Kcnj16 or other molecular players involved in the direct adaptation of ENaC and ROMK function to altered plasma K + concentrations.…”

Role and mechanisms of regulation of the basolateral Kir4.1/Kir5.1K+channels in the distal tubules

“…The L166Q variant reduced channel function and the Q212R mutant did not reduce overall conductance, but did demonstrate subtle effects on spermine sensitivity (Méndez‐González et al . ). Tanemoto et al .…”

“…As described by Méndez González et al . (), there are over 120 coding‐region single nucleotide polymorphisms (SNPs) in the KCNJ10 gene reported in the publicly accessible genome databases. Electrophysiological analysis revealed that the currents produced by mutant channels carrying disease‐associated mutations (R65P, R65P/R199X, G77R, C140R, T164I and A167V/R297C) were strongly reduced, indicating that the loss of channel function underlies the EAST/SeSAME syndrome (Williams et al .…”

Exploring Neuronal Vulnerability to Head Trauma Using a Whole Exome Approach

“…We found a rare predicted-deleterious variant in KCNJ10, a gene previously implicated in symptom development and recovery following mTBI. 53 KCNJ10 encodes the potassium channel Kir4.1, an inwardly rectifying potassium channel, which restores negative resting potential mediating the clearing of extracellular glutamate. 54 The case patient (R117) identified with a KCNJ10 variant (p.Arg18Trp) presented with recurrent episodes of pallor and vomiting following minor head injuries.…”