Increasing the medium osmolality, with a non‐ionic osmoticant, from control (289 mOsm) to 319 mOsm or 344 mOsm in the lumbrical muscle cell of the mouse, resulted in a depolarization of the membrane potential (Vm) of 5.9 mV and 10.9 mV, respectively. In control medium, the blockers of chloride related cotransport bumetanide and furosemide, induced a hyperpolarization of −3.6 and −3.0 mV and prevented the depolarization due to hypertonicity. When bumetanide was added in hypertonic media Vm fully repolarized to control values. In a medium of 266 mOsm, the hyperpolarization by bumetanide was absent. At 344 mOsm the half‐maximal effective concentration (IC50) was 0.5 μm for bumetanide and 21 μm for furosemide. In solutions containing 1.25 mm sodium the depolarization by hypertonicity was reduced to 2.3 mV. Reducing chloride permeability, by anthracene 9 carboxylic acid (9‐AC) in 289 mOsm, induced a small but significant hyperpolarization of −2.6 mV. Increasing medium osmolality to 344 mOsm enlarged this hyperpolarization significantly to −7.6 mV. In a solution of 344 mOsm containing 100 μm ouabain, the bumetanide‐induced hyperpolarization of Vm was absent. The results indicate that a Na‐K‐2Cl cotransporter is present in mouse lumbrical muscle fibre and that its contribution to Vm is dependent on medium osmolality. British Journal of Pharmacology (1997) 120, 39–44; doi:

In resting skeletal muscle the potassium permeability is determined by the permeability of the inwardly potassium rectifier. Continuous resting membrane potential measurements are done to follow the relaxation of the membrane potential upon changes in potassium permeability. Inhibition of the inwardly potassium rectifier, by extracellular application of 80 microM Ba(2+), causes the cell to depolarize with mean time constants as follows: in control 127+/-7 s ( n=23), in the presence of bumetanide, as an inhibitor of the Na(+)/K(+)/2Cl(-) cotransporter, 182+/-23 s ( n=7), in hypertonic media (340 mosmol/kg) 90.4+/-5 s ( n=7) and in reduced chloride medium 64+/-8 s ( n=5). The depolarizing relaxation of the membrane potential induced by reduction of extracellular potassium produces similar results. These time constants are at least three orders of magnitude slower than the time constants reported in the literature for the inhibition of the inwardly potassium rectifier. Chloride transport affects the relaxation of the membrane potential. A further characterization of chloride transport is done by following the relaxation of the membrane potential upon application of chloride transport modulators. It is argued that the electroneutral cotransporter, for which a flux was preliminarily estimated of 13.4 pmol cm(-2) s(-1), has a considerable role in the processes related to the resting membrane potential.

The lumbrical skeletal muscle fibres of mice exhibited electrically bistable behaviour due to the nonlinear properties of the inwardly rectifying potassium conductance. When the membrane potential (Vm) was measured continuously using intracellular microelectrodes, either a depolarization or a hyperpolarization was observed following reduction of the extracellular potassium concentration (K+o) from 5.7 mm to values in the range 0.76–3.8 mm, and Vm showed hysteresis when K+o was slowly decreased and then increased within this range. Hypertonicity caused membrane depolarization by enhancing chloride import through the Na+–K+–2Cl− cotransporter and altered the bistable behaviour of the muscle fibres. Addition of bumetanide, a potent inhibitor of the Na+‐K+‐2Cl− cotransporter, and of anthracene‐9‐carboxylic acid, a blocker of chloride channels, caused membrane hyperpolarization particularly under hypertonic conditions, and also altered the bistable behaviour of the cells. Hysteresis loops shifted with hypertonicity to higher K+o values and with bumetanide to lower values. The addition of 80 μM BaCl2 or temperature reduction from 35 to 27 °C induced a depolarization of cells that were originally hyperpolarized. In the K+o range of 5.7–22.8 mm, cells in isotonic media (289 mmol kg−1) responded nearly Nernstianly to K+o reduction, i.e. 50 mV per decade; in hypertonic media this dependence was reduced to 36 mV per decade (319 mmol kg−1) or to 31 mV per decade (340 mmol kg−1). Our data can explain apparent discrepancies in ΔVm found in the literature. We conclude that chloride import through the Na+–K+–2Cl− cotransporter and export through Cl− channels influenced the Vm and the bistable behaviour of mammalian skeletal muscle cells. The possible implication of this bistable behaviour in hypokalaemic periodic paralysis is discussed.

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