H.G.J. van Mil

Effects of chloride transport on bistable behaviour of the membrane potential in mouse skeletal muscle

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.

The influence of bumetanide on the membrane potential of mouse skeletal muscle cells in isotonic and hypertonic media

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.

Modulation of the isoprenaline-induced membrane hyperpolarization of mouse skeletal muscle cells.

1 The hyperpolarization of the resting membrane potential, Vm, induced by isoprenaline in the lumbrical muscle fibres of the mouse, was investigated by use of intracellular microelectrodes. 2 In normal Krebs‐Henseleit solution (potassium concentration: K+o=5.7mM, ‘control’), Vm was −74.0 ± 0.2 mV; lowering K+o to 0.76 mM (′low K+o) resulted in either a hyperpolarization (Vm= −95.7 ±2.9 mV), or a depolarization (Vm= −52.0 ±0.3 mV). 3 Isoprenaline (≥200 nM) induced a hyperpolarization of Vm by ΔVm= − 5.6 ±0.4 mV in control solution. 4 When Vm hyperpolarized after switching to low K+o, the addition of isoprenaline resulted in increased hyperpolarization Vm: ΔVm=−16.3±3.2 mV to a final Vm=−110.1 ±3.4 mV. Adding iso‐prenaline when Vm depolarized in low K+o, leads to a hyperpolarization of either by − 11.6±0.5mV to −63.6 ±0.8 mV or by −51.7 ±2.7 mV to −106.9 ±3.9 mV. 5 Ouabain (0.1 to 1 mm) did not suppress the hyperpolarization by isoprenaline in 5.7 mM K+o (ΔVm=−6.7±0.4mV) or the hyperpolarization of the depolarized cells in low K+o (ΔVm=−9.7 Δ1.5mV). 6 The hyperpolarization is a logarithmically decreasing function of K+o in the range between 2 and 20 mM (12 mV/decade). 7 IBMX and 8Br‐cyclic AMP mimicked the response to isoprenaline whereas forskolin (FSK) induced in low K+o a hyperpolarization of −7.0 ±0.7 mV that could be augmented by addition of isoprenaline (ΔVm=−8.2±1.8mV). 8 In control and low K+o, Ba2+ (0.6 mm) inhibited the hyperpolarization induced by isoprenaline, IBMX or 8Br‐cyclic AMP. Other blockers of the potassium conductance such as TEA (5 mm) and apamin (0.4 μm) had no effect. 9 We conclude that in the lumbrical muscle of the mouse the isoprenaline‐induced hyperpolarization is primarily due to an increase in potassium permeability.

Osmolality influences bistability of membrane potential under hypokalemic conditions in mouse skeletal muscle: an experimental and theoretical study.

Abstract The membrane potential in mouse skeletal muscle depends on both extracellular osmolality and potassium concentration. These dependencies have been related to two membrane transporters, Na + /K + /2Cl − co-transporter and the inward potassium rectifier channel. To investigate the relation of the Na + /K + /2Cl − co-transporter and the inward potassium rectifier channel in a qualitative way, a combined electrophysiological and modelling approach was used. The experimental results show that the bistability of the membrane potential, which is related to the conductive state of the inward potassium rectifier channel, is shifted to higher extracellular potassium values when medium osmolality is increased. These results are confirmed by the computer simulation calculations for increased co-transporter flux. The combined results indicate that the co-transporter is capable of modulating the conductive state of the inward potassium rectifier channel.