A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation

Abstract

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K ؉ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by G ␤␥ subunits, but instead shows constitutive activation, and (ii) is no longer a K ؉ -selective channel but conducts Na ؉ as well. The present experiments on weaverGIRK2 (wv-GIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na ؉ concentration. In particular, manipulations that decrease intracellular Na ؉ produce a component of Na ؉ -permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na ؉ inf lux through the wvGIRK2 channel, leading to additional Na ؉ activation. We also show that the wvGIRK2 channel is permeable to Ca 2؉ , providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K ؉ channels.The weaver mouse carries the first vertebrate ion channel mutation associated with a developmental defect. The singlenucleotide mutation changes a glycine residue to serine in the pore region of the G protein-gated inward rectifier K ϩ channel (GIRK2), altering the highly conserved and critical GYG sequence to SYG (1). In homozygotic weaver mice, the mutation causes degeneration of the cerebellar granule cells during migration from the external germinal layer to the granule cell layer (2-4), degeneration of the substantia nigra pars compacta (5), cytoarchitectonic anomalies in the CA3 region of the hippocampus (6), seizures, and male sterility.How does the weaver mutation cause these defects? Previous studies showed that the weaverGIRK2 (wvGIRK2) channel expressed in heterologous systems displays three new functional properties: (i) wvGIRK2 no longer requires activation by G ␤␥ subunits, but instead shows constitutive activation; (ii) wvGIRK2 is no longer selective for K ϩ , but conducts Na ϩ as well; and (iii) wvGIRK2 is sensitive to blockade by several drugs, including QX-314, MK-801, and verapamil, that block other cation channels (7-10). In a previous study (8), this drug sensitivity was exploited: when weaver granule cells were incubated in culture with cation channel blockers, they survived, extended neurites, and displayed a marker of differentiation. The degenerative weaver phenotype is explained by the straightforward hypothesis that Na ϩ influx through the mutant channel eventually overloads the Na ϩ extrusion capabilities of the granule cell, leading to collapse of the Na ϩ gradient, the resulting collapse...

Hyperinsulinism induced by targeted suppression of beta cell K ATP channels

“…Whereas fura-4F fluorescence is largely targeted to the cytosol, cytosolic fluorescence of EGFP-tagged K ATP channels, measured separately under the same conditions, was relatively low (J.D.J., unpublished observations). [4][5][6][7][8][9][10][11][12][13][14], there was also no difference in serum insulin levels between AAA-TG and control littermates (8). However, analysis of serum insulin levels of fasted and fed adult mice (4-8 months) indicated consistently 2.5-fold higher levels in AAA-TG than in control littermates (Table 1, Fig.…”

“…Miki et al (5) generated mice expressing a dominant-negative Kir6.2 mutant (Kir6.2[G132S]) in pancreatic beta cells under insulin promoter control. The G132S mutation perturbs the structure of the K ϩ -selectivity filter of the channel and renders channels nonfunctional or, perhaps, slightly Na ϩ -permeable (10). Beta cells from these transgenic mice show reduced K ATP currents, accompanied by a significant increase in both the resting membrane potential and [Ca 2ϩ ] i (5).…”

A Cell Cycle Alteration Precedes Apoptosis of Granule Cell Precursors in the Weaver Mouse Cerebellum

“…[5][6][7][8][9][10][11][12] It is generally assumed that target cells in weaver mice die as a result of chronic depolarization; however, the pathogenetic mechanisms that lead to selective activation of apoptosis in cerebellar granule cell precursors but not in dopaminergic neurons of midbrain are not completely understood. Here we show that abnormalities in the control of cell cycle in wv/wv cerebellum represent a major and early consequence of the altered GIRK2 channel function that may strongly influence the susceptibility of EGL cells to undergo apoptosis.…”

“…4 In vitro, the mutation leads to a complex series of changes in channel activity, ranging from a loss of G-proteindependent inward rectifier current regulation 5,6 to reduced selectivity for potassium over sodium and calcium ions. [7][8][9][10][11][12] This alteration results in chronic depolarization and cell death. In vivo studies show that the granule cell precursors of the external germinal layer (EGL) of the cerebellum die massively via apoptosis within the first 3 postnatal weeks.…”

Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K + channels

“…Ethanol may not activate ROMK (Kir1) and IRK (Kir2) channels because of their high affinity for PIP 2 . The model may also explain the ethanol insensitivity of GIRK2 wv channels (22); the enhanced Na ϩ activation of GIRK2 wv channels may stabilize PIP 2 , thereby decreasing ethanol activation (53)(54)(55).…”