ABSTRACT
Title
Biophysical and pharmacological characterization of skeletal muscle F167L and G190S hClC-1 mutations responsible for myotonia congenita
Authors
G. Gramegna1, J.-F. Desaphy1, M.M. Dinardo1, A. Modoni2, E.M. Valente3, M. Lo Monaco2, D. Conte Camerino1.
(1) Section of Pharmacology, Department of Pharmacobiology, University of "Aldo Moro", Bari
(2) Department of Neuroscience, Catholic University, Rome
(3) IRCCS CSS-Mendel Institute, Roma and University of Messina
(1) Section of Pharmacology, Department of Pharmacobiology, University of "Aldo Moro", Bari
(2) Department of Neuroscience, Catholic University, Rome
(3) IRCCS CSS-Mendel Institute, Roma and University of Messina
Abstract
Myotonia congenita (MC) is an inherited skeletal muscle disease characterized by muscle stiffness during sudden forceful movement, due to electrical instability of the sarcolemma. In affected muscle fibers a greatly diminished sarcolemmal chloride conductance is the basis for the myotonic phenotype (Adrian & Bryant, 1974). MC is a channelopathy due to loss-of-function mutations in CLCN1 gene coding for the skeletal muscle ClC-1 chloride channels. Patient condition was recently worsened by the market withdrawal of the sodium channel blocker mexiletine, the first-choice drug in MC. Acetazolamide (ACTZ), a carbonic anhydrase inhibitor used empirically by MC patients, might increase recombinant hClC-1 activity (Eguchi et al, 2006).
We studied two CLCN1 mutations, F167L and G190S, which were found in an MC patient database and associated steadily with the presence (G190S) or the absence (F167L) of transient weakness (Modoni et al, 2011).
Mutations were introduced in hClC-1 using standard two-step PCR-based site-directed mutagenesis. The hClC-1 mutants and wild type (WT) channels were expressed in HEK 293 cells and chloride currents were measured using the whole-cell patch-clamp technique.
Whatever the chloride ion concentration in the cells, F167L induced little change in chloride currents with respect to WT. Unexpectedly, the current density of F167L was slightly increased in both instantaneous and stationary currents respect to WT. The voltage-dependence of open probability (Popen) was not significantly shifted by about +2 mV in F167L channels. Also the deactivation kinetics of the slow and fast gates of ClC-1 channels were not affected by the mutation.
In contrast, the G190S mutation determines a profound alteration of chloride currents due to a dramatic positive shift of the Popen voltage-dependence; the half-maximum potential of the Popen curve resulted +73 ± 11 mV compared to -30.3 ± 0.6 mV for WT.
The drug ACTZ (300 µM) was tested on WT and G190S hClC-1 currents using various internal HEPES concentrations. In all tested conditions, the drug displayed only a marginal effect on both WT and G190S channels, consisting in a very slight increase at negative potential.
In conclusion, the F167L mutation has little effect on chloride current, leaving unexplained the genotype/phenotype relationship; the G190S mutation dramatically reduce chloride currents, which likely explains myotonia; ACTZ does not significantly improve the currents sustained by WT or G190S channels, suggesting another mechanism of action involved in its antimyotonic activity.
Supported by Telethon-Italy (grant GGP10101) and the “Association Française contre les Myopathies”.
Adrian & Bryant (1974). J Physiol. 240(2): 505-515.
Eguchi et al. (2006). Muscle Nerve. 34(3): 292-297.
Modoni et al. (2011). J Clin Neurophysiol. 28: 39-44.
We studied two CLCN1 mutations, F167L and G190S, which were found in an MC patient database and associated steadily with the presence (G190S) or the absence (F167L) of transient weakness (Modoni et al, 2011).
Mutations were introduced in hClC-1 using standard two-step PCR-based site-directed mutagenesis. The hClC-1 mutants and wild type (WT) channels were expressed in HEK 293 cells and chloride currents were measured using the whole-cell patch-clamp technique.
Whatever the chloride ion concentration in the cells, F167L induced little change in chloride currents with respect to WT. Unexpectedly, the current density of F167L was slightly increased in both instantaneous and stationary currents respect to WT. The voltage-dependence of open probability (Popen) was not significantly shifted by about +2 mV in F167L channels. Also the deactivation kinetics of the slow and fast gates of ClC-1 channels were not affected by the mutation.
In contrast, the G190S mutation determines a profound alteration of chloride currents due to a dramatic positive shift of the Popen voltage-dependence; the half-maximum potential of the Popen curve resulted +73 ± 11 mV compared to -30.3 ± 0.6 mV for WT.
The drug ACTZ (300 µM) was tested on WT and G190S hClC-1 currents using various internal HEPES concentrations. In all tested conditions, the drug displayed only a marginal effect on both WT and G190S channels, consisting in a very slight increase at negative potential.
In conclusion, the F167L mutation has little effect on chloride current, leaving unexplained the genotype/phenotype relationship; the G190S mutation dramatically reduce chloride currents, which likely explains myotonia; ACTZ does not significantly improve the currents sustained by WT or G190S channels, suggesting another mechanism of action involved in its antimyotonic activity.
Supported by Telethon-Italy (grant GGP10101) and the “Association Française contre les Myopathies”.
Adrian & Bryant (1974). J Physiol. 240(2): 505-515.
Eguchi et al. (2006). Muscle Nerve. 34(3): 292-297.
Modoni et al. (2011). J Clin Neurophysiol. 28: 39-44.