Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle

Abstract

1. Ion-sensitive electrodes (made with a chloride-sensitive ion-exchange resin) were used to measure the internal chloride activity (a_i^Cl) of frog sartorius fibres at 25° C.

2. The internal pH (pH_i) of other sartorius fibres was measured with a recessed tip pH-sensitive electrode (made with pH-sensitive glass).

3. In normal bicarbonate-free solution (containing 2·5 mM potassium), the average chloride equilibrium potential, E_Cl (calculated from a_i^Cl and the measured chloride activity of the external solution (a_o^Cl) was 87·7 ± 1·7 mV (mean ± S.E.; n = 16) in fibres where the average membrane potential, E_m, was 88·3 ± 1·5 mV (mean ± S.E.; n = 16). In experiments where a_i^Cl was varied between about 1 and 10 mM (which corresponds to values of E_m between about -105 and -50 mV) E_Cl was within 1-3 mV of E_m at equilibrium. These measurements of a_i^Cl were obtained from the potential difference between the chloride-sensitive electrode and an intracellular indifferent micro-electrode filled with potassium chloride. If a potassium sulphate-filled indifferent micro-electrode was used, then values of a_i^Cl below about 5 mM were erroneously high, probably due to interference from other sarcoplasmic ions at the indifferent electrode.

4. In solutions containing 15 mM bicarbonate and gassed with 5% CO₂, pH_i was 6·9, corresponding to an internal bicarbonate concentration of 7·6 mM. E_Cl measured in this solution was some 4 mV positive to E_m. Most of the difference between E_Cl and E_m could be ascribed to interference by sarcoplasmic bicarbonate on the basis of selectivity measurements of chloride against bicarbonate made on the ion-exchange resin in the relevant range of a^Cl.

5. If bicarbonate/CO₂ in the external solution was replaced by HEPES/pure O₂ at constant pH, then pH_i rose from 6·88 ± 0·02 (mean ± S.E.) to 7·05 ± 0·02. A change in external pH of 1 unit caused pH_i to change by about 0·02 unit and the intracellular buffering power was calculated to be about 35.

6. In solution made hypertonic by the addition of sucrose, E_m changed little or depolarized and E_Cl and E_m remained close. In contrast, in solution made hypertonic by the addition of solid sodium chloride (high-chloride solution) E_Cl became negative to E_m. Conversely in low chloride solution E_Cl became positive to E_m.

7. When the chloride permeability (P_Cl) was reduced by the use of acid solution, E_Cl moved positive to E_m indicating an accumulation of internal chloride. When P_Cl was increased again by returning to more alkaline solution, E_m depolarized to E_Cl.

8. The results are consistent with the existence of a small, active movement of chloride, the effects of which are normally obscured by large passive movements of chloride when P_Cl is large.

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