Elevated resting H+ current in the R1239H type 1 hypokalaemic periodic paralysis mutated Ca2+ channel

Abstract

Key points: Missense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage-gated Ca²⁺ channel induce type 1 hypokalaemic periodic paralysis, a poorly understood neuromuscular disease characterized by episodic attacks of paralysis associated with low serum K⁺ . Acute expression of human wild-type and R1239H HypoPP1 mutant α1 subunits in mature mouse muscles showed that R1239H fibres displayed Ca²⁺ currents of reduced amplitude and larger resting leak inward current increased by external acidification. External acidification also produced intracellular acidification at a higher rate in R1239H fibres and inhibited inward rectifier K⁺ currents. These data suggest that the R1239H mutation induces an elevated leak H⁺ current at rest flowing through a gating pore and could explain why paralytic attacks preferentially occur during the recovery period following muscle exercise.

Abstract: Missense mutations in the gene encoding the α1 subunit of the skeletal muscle voltage-gated Ca²⁺ channel induce type 1 hypokalaemic periodic paralysis, a poorly understood neuromuscular disease characterized by episodic attacks of paralysis associated with low serum K⁺ . The present study aimed at identifying the changes in muscle fibre electrical properties induced by acute expression of the R1239H hypokalaemic periodic paralysis human mutant α1 subunit of Ca²⁺ channels in a mature muscle environment to better understand the pathophysiological mechanisms involved in this disorder. We transferred genes encoding wild-type and R1239H mutant human Ca²⁺ channels into hindlimb mouse muscle by electroporation and combined voltage-clamp and intracellular pH measurements on enzymatically dissociated single muscle fibres. As compared to fibres expressing wild-type α1 subunits, R1239H mutant-expressing fibres displayed Ca²⁺ currents of reduced amplitude and a higher resting leak inward current that was increased by external acidification. External acidification also produced intracellular acidification at a higher rate in R1239H fibres and inhibited inward rectifier K⁺ currents. These data indicate that the R1239H mutation induces an elevated leak H⁺ current at rest flowing through a gating pore created by the mutation and that external acidification favours onset of muscle paralysis by potentiating H⁺ depolarizing currents and inhibiting resting inward rectifier K⁺ currents. Our results could thus explain why paralytic attacks preferentially occur during the recovery period following intense muscle exercise.

Keywords: gating pore; myopathy; voltage-gated Ca2+ channel.

Figure 1. Calibration of the pH‐sensitive indicator BCECF

A, recording of the changes in BCECF fluorescence ratio in a WT fibre held at −80 mV in response to acidification of the external Tyrode solution and exposure to the calibration solutions. After challenging the cell with the pH 5 buffered external solution and returning to pH 7.2, the external solution was slowly exchanged for a 140 mm K‐containing solution with 10 μm nigericin added and buffered at pH 7, then 8 and finally 5.5. This protocol was applied in each WT and R1239H fibre tested. B, recording of the corresponding changes in pH_i induced by acidification of the external solution in the same fibre as in A after conversion of the fluorescence ratios into pH values using the 3 fluorescence ratios measured in the presence of the 3 calibrating solutions.

Figure 2. Distribution of GFP‐tagged human WT and R1239H α1 subunits in adult mouse skeletal muscle fibres

A and B, transmitted light images of fibres expressing WT and R1239H α1 subunits. C and D, corresponding confocal images of GFP fluorescence in the fibres presented in A and B. E and F, fluorescence intensity profiles from the white box regions in C and D, respectively.

Figure 3. L‐type voltage‐gated Ca²⁺ currents in WT and R1239H fibres

A, recordings of L‐type currents (upper traces) in a WT and in an R1239H fibre in response to depolarizing pulses of 1 s duration to the indicated voltages (lower traces). B, relationships between the mean peak values of L‐type Ca²⁺ currents and membrane voltage in the two fibre types. C, mean of the fitting parameters of current–voltage relationships obtained in each WT and R1239H fibre.

Figure 4. Leak currents and leak conductance in WT and R1239H fibres

A, means and SEM of current densities evoked by voltage ramps applied from a holding potential of 0 mV in WT and R1239H fibres. The number of data points has been reduced for clarity. B, mean slope membrane conductance measured between −120 and −80 mV for voltage ramp‐evoked membrane currents in the two fibre types from different holding potentials (HP). The number of fibres tested is indicated above each histogram bar.

Figure 5. Effect of external acidification on leak currents in WT and R1239H fibres

A, currents evoked by voltage ramps applied from a holding potential of 0 mV at an external pH of 7.2 and 6 and current difference in the same R1239H fibre. B, means and SEM of current densities evoked by voltage ramps applied from a holding potential of 0 mV at an external pH of 7.2 and 6 in R1239H fibres. C, means and SEM of current densities evoked by voltage ramps applied from a holding potential of 0 mV at an external pH of 7.2 and 6 in WT fibres. D, mean differences and SEM between currents evoked by voltage ramps applied from a holding potential of 0 mV at pH 6 and at pH 7.2 in WT and in R1239H fibres. The number of data points has been reduced for clarity.

Figure 6. Effect of external acidification on BCECF fluorescence and leak currents in WT and R1239H fibres in the presence of a TEA‐MeSO₃‐containing external solution

A, simultaneous recording of BCECF fluorescence ratio (upper trace) and membrane currents (lower trace) in an R1239H muscle fibre held at −80 mV and stimulated by 50 ms duration voltage pulses given to −90 mV at a frequency of 0.5 Hz. The background current at −80 mV has been zoomed so that the downward deflections of the currents evoked by hyperpolarizing pulses are not presented in full. B, superimposed representative traces of membrane currents recorded in response to short pulses given to −90 mV just before and 60 s after the change of external pH. C, mean rate of change in BCECF fluorescence ratio during the first minute of exposure of the cell to the external solution buffered at pH 5. The number of fibres tested is indicated above each bar.

Figure 7. Effect of external acidification on pH_i in WT and in R1239H fibres in the presence of an external Tyrode solution

A, mean and SEM of change in pH_i as a function of time in WT and in R1239H fibres. B, mean rate of change in pH_i during the first minute of exposure of the cell to the external solution buffered at pH 5. The number of fibres tested is indicated above each bar.

Figure 8. Effect of external acidification on Kir channels

A, recording of membrane currents in a control muscle fibre held at −80 mV and stimulated by 50 ms duration voltage pulses given to −90 mV at a frequency of 0.5 Hz in the presence of an external Tyrode solution. B, mean and SEM of normalized Kir currents evoked by voltage ramps applied from a holding potential of −40 mV at an external pH of 7.2 and 6 in 6 control fibres. Each cell was exposed to 4 solutions, a Tyrode solution buffered at pH 7.2, a Tyrode solution buffered at pH 6, either with or without 0.5 mm Ba²⁺. In each cell, currents were normalized to the current obtained at −120 mV in the presence of the Tyrode solution buffered at pH 7.2. The two mean current–voltage relationships were obtained, respectively, after the following substractions: the currents obtained at pH 7.2 in the absence of Ba²⁺ minus the currents obtained at pH 7.2 in the presence of Ba²⁺ (○), and the currents obtained at pH 6 in the absence of Ba²⁺ minus the currents obtained at pH 6 in the presence of Ba²⁺ (Δ). C, mean currents measured at −80 mV after normalization to the current obtained at −120 mV in the 4 solutions used and after subtraction. Values were compared using paired t tests.