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

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 m M 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 m M (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 m M were erroneously high, probably due to interference from other sarcoplasmic ions at the indifferent electrode. 4. In solutions containing 15 m M bicarbonate and gassed with 5% CO 2 , pH i was 6·9, corresponding to an internal bicarbonate concentration of 7·6 m M . 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/CO 2 in the external solution was replaced by HEPES /pure O 2 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.