IDENTIFICATION OF FUNCTIONALLY IMPORTANT REGIONS OF THE MUSCULAR CHLORIDE CHANNEL CIC-1 BY ANALYSIS OF RECESSIVE AND DOMINANT MYOTONIC MUTATIONS

Mutations in the muscular voltage-dependent Cl- channel, CIC-1, lead t o recessive and dominant myotonia, Here we analyse the effects of one dominant (G200R) and three recessive (Y150C, Y261C, and M485V) mutatio ns after functional expression in Xenopus oocytes, Glycine 200 is a hi ghly conserved amino acid located in a conserved stretch in the putati vely cytoplasmic loop between domains D2 and D3, Similar to several ot her dominant mutations the amino acid exchange G200R leads to a drasti c shift by similar to 65 mV of the open probability curve to more posi tive voltages, As explored by co-expression studies, the shift is inte rmediate in heteromeric mutant/WT channels, Open channel properties su ch as single channel conductance, rectification or ion selectivity are not changed, Thus we identified a new region of the CIC-1 protein in which mutations can lead to drastic shifts of the voltage dependence, The recessive mutation M485V, which is located in a conserved region a t the beginning of domain D10, leads to a drastic reduction of the sin gle channel conductance from 1.5 pS for WT to similar to 0.3 pS, In ad dition, the mutant is strongly inwardly rectifying and deactivates inc ompletely at negative voltages, ion-selectivity, however, is unchanged , These electrophysiological properties fully explain the recessive ph enotype of the mutation and identify a new region of the protein that is involved in ion permeation and gating of the CIC-1 channel, The oth er two recessive mutations (Y150C and Y261C) had been found in a compo und heterozygous patient, Surprisingly, expression of these mutants in oocytes yielded currents indistinguishable from WT CIC-1 when explore d by two-electrode voltage clamp recording and patch clamping (either singly or both mutations co-expressed), Other mechanisms that are not faithfully represented by the Xenopus expression system must therefore be responsible for the myotonic symptoms associated with these mutati ons.