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Keywords:

  • chloride channel;
  • excitability;
  • myotonia congenita;
  • sex hormones;
  • skeletal muscle

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Introduction: Myotonia congenita, caused by mutations in ClC-1, tends to be more severe in men and is often exacerbated by pregnancy. Methods: We performed whole-cell patch clamp of mouse muscle chloride currents in the absence/presence of 100 μM progesterone or 17β-estradiol. Results: 100 μM progesterone rapidly and reversibly shifted the ClC-1 activation curve of mouse skeletal muscle (V50 changed from −52.6 ± 9.3 to +35.5 ± 6.7; P < 0.01) and markedly reduced chloride currents at depolarized potentials. 17β-estradiol at the same concentration had a similar but smaller effect (V50 change from −57.2 ± 7.6 to −40.5 ± 9.8; P < 0.05). 1 μM progesterone produced no significant effect. Conclusions: Although the data support the existence of a nongenomic mechanism in mammalian skeletal muscle through which sex hormones at high concentration can rapidly modulate ClC-1, the influence of hormones on muscle excitability in vivo remains an open question. Muscle Nerve 48: 589–591, 2013

The voltage-gated chloride channel, ClC-1, is important in regulation of muscle excitability; it matches the properties of a myofiber with the demands of its motor neuron[1] and combats fatigue.[2, 3] A role for sex hormones in muscle excitability is suggested by the observations that the chloride channel disease myotonia congenita tends to be more severe in men and is often exacerbated by pregnancy.[4, 5] In Xenopus oocytes, ClC-1 can be modulated by sex hormones through a rapid, and, therefore, nongenomic, mechanism.[7] We asked whether such a mechanism exists in mammalian skeletal muscle.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Isolated flexor digitorum brevis (FDB) myofibers from mice aged between 1 and 4 months were studied by whole-cell patch clamp following the method of Lueck et al.[6] Briefly, animals were killed by isoflurane inhalation and cervical dislocation, and the FDB muscle from a hindfoot was triturated in a series of 35-mm plastic tissue culture dishes of Ringer solution (in mM: NaCl 146, KCl 5, MgCl2 1, CaCl2 2, HEPES 10 [(4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid], pH 7.4) after incubation for 1 h at 37°C in 1 mg/ml collagenase A dissolved in Ringer solution. Animals were shared with a colleague conducting experiments on brain, some of which harbored a mutation in Kv1.1. In the experiments presented here, Kv channels were blocked by tetraethylammonium (TEA). No differences were observed between mutant and wildtype animals with respect to the hormone sensitivity of chloride conductance.

Data were collected using fire-polished thin glass microelectrodes with an Axopatch 200B, sampling at 10 kHz with a 2 kHz low-pass filter. Series resistance was 1–3 MΩ and was compensated 90%. Recording solutions at pH 7.4 contained in mM, internal: Cs-Aspartate 110, Cs-Cl 30, MgCl2 5, HEPES 10; external: TEA-Cl 145, CaCl2 10, CdCl2 0.25, HEPES 10. Ringer and external solutions was supplemented with 10 μM BTS to reduce contractions. Stock solutions of BTS, progesterone, and 17β-estradiol were made in dimethyl sulfoxide (DMSO). The total concentration of DMSO in the extracellular recording solution did not exceed 0.22%.

Tail currents were measured without leak subtraction at −100 mV after pre-pulses to voltages from −140 to +120 mV. For each cell, the maximal prehormone current was used to normalize both the pre- and posthormone currents.

Pre- and posthormone parameters for progesterone and estrogen were compared in paired t-tests. The effect of progesterone alone, estrogen alone, and progesterone after prior exposure to estrogen were compared in a one-way analysis of variance.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In the presence of 100 μM progesterone the ClC-1 activation curve was shifted in a depolarizing direction (V50 changed from −52.6 ± 9.3 mV to +35.5 ± 6.7 mV, P < 0.01), and peak current was reduced with the overall effect of reducing chloride currents at depolarized potentials (Fig. 1). The 17β-estradiol at the same concentration had a similar but smaller effect (V50 change from −57.2 ± 7.6 mV to −40.5 ± 9.8 mV, P < 0.05). These effects were complete within the time it took to exchange the solutions and re-start recording (minutes) and could be reversed by washing the hormone off with a similar time scale, suggesting a nongenomic mechanism. However, 1 μM progesterone produced no significant effect (data not shown).

image

Figure 1. Comparison of chloride tail currents before and after application of 100 μM 17β-estradiol (left) or progesterone (right) to the bath. Top row: Representative chloride currents before (grey) and after (black) 100 μM hormone. (A) A cell before/after progesterone. (B) A different cell before/after 17β-estradiol. Scale bars show 200 ms horizontally, 20 nA vertically. Middle row: Fraction tail current as a proportion of pre-hormone maximal current. Means ± SEM are displayed with Boltzmann fits. Circles in C and D are before application of hormone. (C) The effect of 100 μM progesterone (triangles, n = 5). (D) The 17β-estradiol was applied first (squares, n = 5) and after washoff, progesterone was applied (triangles; n = 3). Tail currents at 0 mV were significantly smaller in the presence of progesterone or 17β-estradiol (stars, P < 0.01, paired t-tests). Progesterone after prior exposure to 17β-estradiol produced significantly smaller tail currents at 0 mV than either progesterone or 17β-estradiol alone (dot, P < 0.01, one-way analysis of variance and post hoc pairwise analysis). Bottom row: Effects of progesterone (E) and 17β-estradiol (F) are reversed by washing hormone off. Before hormone (open circles), after 100 μM hormone (triangles, squares), wash (closed circles), n = 1.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In Xenopus oocytes sex hormones rapidly reduce the conductance through heterologously expressed human ClC-1[7]; we found that the endogenous chloride conductance in mammalian skeletal muscle responds similarly. The components of the pathway linking sex hormones and ClC-1 remain to be explored. Protein kinase C is known both to regulate ClC-1[8, 9] and to mediate certain nongenomic actions of progesterone in the brain.[10] However, as in the oocyte experiments, the concentration of hormone that was effective in our recordings is much higher than what occurs naturally in blood; serum progesterone levels during late pregnancy are approximately 0.2 μM.[11] Thus, although the data support the existence of a mechanism in skeletal muscle through which sex hormones at high concentration can rapidly modulate ClC-1, the influence of hormones on muscle excitability in vivo remains an open question.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
Abbreviations
DMSO

dimethyl sulfoxide

FDB

flexor digitorum brevis

HEPES

4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid

TEA

tetraethylammonium