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Abstract
Mutations in the muscle chloride channel gene (CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. We here studied the functional consequences of four disease-causing mutations that predict amino acid substitutions Q43R, S70L, Y137D and Q160H. Wild-type (WT) and mutant hClC-1 channels were heterologously expressed as YFP or CFP fusion protein in HEK293T cells and analyzed by whole-cell patch clamp and fluorescence recordings on individual cells. Q43R, Y137D and Q160H, but not S70L reduced macroscopic current amplitudes, but left channel gating and unitary current amplitudes unaffected. We developed a novel assay combining electrophysiological and fluorescence measurements at the single-cell level in order to measure the probability of ion channel surface membrane insertion. With the exception of S70L, all tested mutations significantly reduced the relative number of homodimeric hClC-1 channels in the surface membrane. The strongest effect was seen for Q43R that reduced the surface insertion probability by more than 99% in Q43R homodimeric channels and by 92 ± 3% in heterodimeric WT/Q43R channels compared to homodimeric WT channels. The new method offers a sensitive approach to investigate mutations that were reported to cause channelopathies, but display only minor changes in ion channel function.
Highlights
Mutations in the muscle chloride channel gene (CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement
In a systematic screen of all 23 exons of the CLCN1 gene in seven patients with recessive generalized myotonia (Becker) we found four missense mutations predicting amino acid exchanges Q43R, S70L, Y137D and Q160H
We tested seven patients with typical myotonic symptoms such as muscle stiffness, muscle hypertrophy and myalgia that improved during exercise for mutations in CLCN1
Summary
Mutations in the muscle chloride channel gene (CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. Skeletal muscle fibers are unique among excitable tissue in a large resting chloride conductance that results in short electrical length constants of the sarcolemma[4]. This feature is necessary for electrical www.nature.com/scientificreports/. We studied homo- and heterodimeric mutant channels in heterologous expression systems and found that all mutations leave channel function unaffected, but reduce surface membrane insertion. To compare the severity of the trafficking defects caused by the different mutations we developed a novel assay that uses a combination of cellular electrophysiology and fluorescence microscopy This approach permitted accurate quantification of the percentage of homoand heterodimeric channels embedded in the plasma membrane
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