Impaired surface membrane insertion of homo- and heterodimeric human muscle chloride channels carrying amino-terminal myotonia-causing mutations.
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.
Institut für Neurophysiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, Hannover, Germany.
Katharina
K
Ronstedt
Hôpital Pitié-Salpêtrière, 47-83 Boulevard de l'Hôpital,Paris, France.
Damien
D
Sternberg
Abteilung Molekulare Pharmakologie, RWTH Aachen University Templergraben 55, Aachen, Germany.
Silvia
S
Detro-Dassen
Institut für Neurophysiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, Hannover, Germany.
Thomas
T
Gramkow
Institut für Neurophysiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, Hannover, Germany.
Birgit
B
Begemann
Institut für Neurophysiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, Hannover, Germany.
Toni
T
Becher
Institut für Neurophysiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, Hannover, Germany.
Petra
P
Kilian
Institut für Neurophysiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, Hannover, Germany.
Matthias
M
Grieschat
Institute of Complex Systems-Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, Germany.
Jan-Philipp
JP
Machtens
Abteilung Molekulare Pharmakologie, RWTH Aachen University Templergraben 55, Aachen, Germany.
Günther
G
Schmalzing
Institut für Neurophysiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, Hannover, Germany.
Martin
M
Fischer
Institute of Complex Systems-Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, Germany.
Christoph
C
Fahlke
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D057809:HEK293 Cells; D006801:Humans; D009132:Muscles / Q000378:metabolism*; D009154:Mutation; D009222:Myotonia / Q000235:genetics / Q000378:metabolism*; D013499:Surface Properties
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels