PMC

Deactivation kinetics of expressed and native ClC-1 channels. Voltage dependence for the relative contributions of the slow (A), fast (B), and nondeactivating (C) gating components of ClC-1 deactivation in mClC-1–expressing HEK293 cells (squares; n = 12), mClC-1–expressing myotubes (triangles; n = 4), and native FDB fibers obtained from 18–20-d-old WT mice (circles; n = 8). (D) Voltage dependence of the time constants for the fast (lower symbols) and slow (upper symbols) components of ClC-1 deactivation. *, P ≤ 0.05 HEK293 cells compared with WT FDB fibers.

Macroscopic properties of expressed and native ClC-1 currents. Voltage dependence of average instantaneous (open symbols) and steady-state (closed symbols) ClC-1 currents recorded from mClC-1–expressing HEK293 cells (A; n = 12), mClC-1–expressing skeletal myotubes (B; n = 4), and native FDB fibers obtained from 18–20-d-old WT mice (C; n = 8). Instantaneous currents measured in control noninjected myotubes did not display appreciable ClC-1 currents (upright triangles). (D) Superimposed and normalized instantaneous current–voltage relationships obtained from mClC-1– expressing HEK293 cells (squares), myotubes (inverted triangles), and native WT FDB fibers (circles). (E) Average relative Po-V curves for mClC-1–expressing HEK293 cells (squares), myotubes (inverted triangles), and native WT FDB fibers (circles) obtained from tail currents elicited at −100 mV. Smooth curves through each P_O-V dataset were generated using a modified Boltzmann equation ().

Nonstationary noise analysis during ClC-1 deactivation in FDB fibers obtained from wild-type and HSA ^LR mice. (A) Representative mean currents in FDB fibers obtained from 12-d-old WT (left) and 18-d-old HSA ^LR (right) mice elicited using a 200-ms voltage step to −140 mV after a 100-ms prepulse to +60 mV. The initial 2 ms of each voltage step is blanked and the total duration of the traces truncated to 400 ms for clarity. (B) Variance time course for the currents shown in A. (C) Mean–variance relationships for the experiments shown in A and fit by (see for average values of fitted parameters).

Deactivation kinetics of ClC-1 currents in FDB fibers obtained from WT and HSA ^LR mice. (A) Superimposed traces of normalized ClC-1 current deactivation (solid lines) elicited at −100 mV in FDB fibers obtained from 19-d-old WT and HSA ^LR mice and fit with a second order exponential (left; white dashed lines). ClC-1 currents from representative WT (middle) and HSA ^LR (right) FDB fibers fit with a biexponential function and replotted on log-linear plots. Fractional amplitudes of the extracted slow, fast, and nondeactivating components of the currents are shown for clarity. (B–D) Voltage dependence of the average relative contribution of the slow (B), fast (C), and steady-state (D) components of ClC-1 deactivation in FDB fibers obtained from 18–20-d-old WT (open circles, n = 8) and HSA ^LR (open squares, n = 16) mice. (E) Voltage dependence of the fast and slow time constants of ClC-1 deactivation in FDB fibers obtained from 18–20-d-old WT (open circles, n = 8) and HSA ^LR (open squares, n = 16) mice. *, P ≤ 0.05.

Macroscopic ClC-1 current density is reduced in FDB fibers from Mbnl1 ^ΔE3/ΔE3 mice. (A) Representative family of ClC-1 currents recorded from FDB fibers obtained from 14-d-old WT (left) and Mbn1l ^ΔE3/ΔE3 (right) mice. (B) Average instantaneous ClC-1 current–voltage curves for fibers obtained from 9–14-d-old WT (closed circles, n = 22) and Mbnl1 ^ΔE3/ΔE3 (open triangles, n = 16) mice. (C) Average relative Po-V curves for FDB fibers obtained from 9–14-d-old WT (closed circles) and Mbnl1 ^ΔE3/ΔE3 (open triangles) mice. Smooth curves through each P_O-V dataset were generated using a modified Boltzmann equation (). (D and E) Representative mean–variance relationships for FDB fibers obtained from 12-d-old WT (D) and Mbnl1 ^ΔE3/ΔE3 (E) mice elicited using a 200-ms voltage step to −140 mV after a 100-ms prepulse to +60 mV (see for average values of fitted parameters).

Macroscopic ClC-1 current density is dramatically reduced in FDB fibers of HSA ^LR mice. Representative family of ClC-1 currents recorded from FDB fibers of 19-d-old WT (A) and HSA ^LR (B) mice elicited using an identical voltage protocol (see , top). The dashed lines represent the zero current level. (C) Voltage dependence of average instantaneous current–voltage relationship in FDB fibers obtained from 18–20-d-old WT (closed circles; n = 8) and HSA ^LR (open squares; n = 16) mice. The apparent depolarizing shift in the reversal potential of the instantaneous ClC-1 current in FDB fibers from HSA^LR mice most likely arises from difficulty in resolving the true reversal potential in face of small, nearly undetectable, outward currents in these fibers. (D) Average relative Po-V curves for FDB fibers obtained from 18–20-d-old WT (closed circles; n = 8) and HSA ^LR (open squares; n = 16) mice. Smooth curves through each P_O-V dataset were generated using a modified Boltzmann equation ().

Chloride channel currents from mClC-1–expressing HEK293 cells, mClC-1–expressing mouse skeletal myotubes, and native FDB fibers obtained from 18–20-d-old WT mice. Representative whole-cell currents recorded first in the absence (Raw) and then in the presence of 1 mM 9AC (9AC) using an identical voltage protocol (top). ClC-1 currents were then quantified after offline subtraction of 9AC-insensitive currents from raw currents (9AC sensitive). (Inset) Capacitative current recorded from the WT FDB fiber resulting from the average of five 10-mV depolarizations delivered from a −80-mV holding potential. The time constant the capacitative current relaxation was fitted (thick line) to a first-order exponential function (τ_m = 339 μs). Cell capacitance and access resistance deduced from the fit were 671 pF and 509 kΩ, respectively. The scale bars for the inset are 6 nA (vertical) and 2 ms (horizontal). The dashed lines represent the zero current level.

Proposed molecular model for increased muscle excitability in DM1. (Left) Proper CLCN1 pre-mRNA splicing in normal skeletal muscle is regulated by muscleblind-like 1 (MBNL1) proteins, which are shown to bind to hypothetical intronic splice repressor elements in intron 6. (Right) Altered CLCN1 pre-mRNA splicing in DM1 arises from DMPK transcripts with expanded repeats (CUG)_n accumulating in nuclear foci and sequestering double-stranded CUG binding proteins, including MBNL1. Depletion of MBNL1 proteins results in inclusion of additional exons (e.g., exon 7a) containing premature termination codons. Additionally, aberrantly spliced CLCN1 transcripts are subsequently exported from the nucleus, degraded through the nonsense-mediated decay pathway, and/or produce truncated proteins that potentially exert additional dominant-negative effects on ClC-1 function (). These effects result in a dramatic reduction in the number of functional ClC-1 channels and a subsequent increase in muscle excitability.

Kinetics of ClC-1 deactivation after prepulse-dependent current reduction. (A) Representative family of ClC-1 currents (at−100 mV) recorded from an FDB fiber obtained from a 19-d-old WT mouse after 200-ms prepulses to potentials ranging from +60 to −140 mV (in 10-mV increments). (B) Voltage dependence of average time-dependent currents (A_fast+A_slow) after prepulse-dependent current reduction. (Inset) Normalized ClC-1 currents (solid lines) fit with a second order exponential in control (60 mV prepulse; circles) and after ∼50% (average = 48.8 ± 0.1%, n = 8) reduction in current magnitude (−70 mV prepulse; squares). Black dashed line indicates the zero current level. (C, bottom) Voltage dependence of the average relative contribution of the fast (closed circles) and slow (open circles) components of ClC-1 deactivation (C, lower) and their respective time constants (C, top). (D) Average relative contribution of the fast and slow components of the time-dependent current (A_fast+A_slow) and their respective time constants to ClC-1 deactivation (at −100 mV) in FDB fibers obtained from WT mice (+60 mV prepulse; black, n = 8), WT mice after ∼50% reduction in ClC-1 current magnitude (−70 mV prepulse; red, n = 8), HSA ^LR mice (+60 mV prepulse; green, n = 14), Mbnl1 ^ΔE3/ΔE3 mice (+60 mV prepulse; white; n = 11). *, P ≤ 0.05 compared with WT +60 mV prepulse.