Human Molecular Genetics, Vol 6, 805-811, Copyright © 1997 by Oxford University Press
B Wollnik, C Kubisch, K Steinmeyer and M Pusch
Mutations in the muscular voltage-dependent Cl-channel, CIC-1, lead to
recessive and dominant myotonia. Here we analyse the effects of one
dominant (G200R) and three recessive (Y150C, Y261C, and M485V) mutations
after functional expression in Xenopus oocytes. Glycine 200 is a highly
conserved amino acid located in a conserved stretch in the putatively
cytoplasmic loop between domains D2 and D3. Similar to several other
dominant mutations the amino acid exchange G200R leads to a drastic shift
by approximately 65 mV of the open probability curve to more positive
voltages. As explored by co-expression studies, the shift is intermediate
in heteromeric mutant/WT channels. Open channel properties such 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 at the beginning of domain
D10, leads to a drastic reduction of the single channel conductance from
1.5 pS for WT to approximately 0.3 pS. In addition, the mutant is strongly
inwardly rectifying and deactivates incompletely at negative voltages. Ion-
selectivity, however, is unchanged. These electrophysiological properties
fully explain the recessive phenotype 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 other two recessive mutations (Y150C and Y261C) had been
found in a compound heterozygous patient. Surprisingly, expression of these
mutants in oocytes yielded currents indistinguishable from WT CIC-1 when
explored 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 mutations.
ARTICLES
Identification of functionally important regions of the muscular chloride channel CIC-1 by analysis of recessive and dominant myotonic mutations
Center for Molecular Neurobiology (ZMNH), Hamburg University, Germany.
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