Background: 

Skeletal muscle expresses an active isoform of the Na⁺/K⁺/2 Cl^--cotransporter (NKCC). The NKCC is activated by an increase in extracellular osmolality. It mediates a secondary active Cl- inward flux. In muscle cells an increased chloride transport rate leads mainly to depolarization, but not to regulatory volume increase [1] as in most other cell types. This is due to the high chloride conductance of muscle fibers. Previous studies have shown a linear relationship between osmolality and membrane potential (e. g. [1]), which is contradictory to the fact, that the transport rate should saturate, when the NKCC is activated to its maximum. The aim of this study was to determine the activation curve of the NKCC activity of mammalian skeletal muscle.

Methods and Results: 

We measured membrane potentials of rat diaphragm and flexor digitorum brevis (FDB) muscles at different extracellular osmolalities (280 – 360 mOsmol/kg). Muscle preparations were incubated in a modified Bretag solution. Osmolality was adjusted by adding appropriate amounts of sucrose. Histogram plots of membrane potentials revealed a bimodal distribution: one population with high (HP) and one with low (LP) membrane potentials. The LP fraction increased with raising osmolalities. The mean membrane potentials were determined after smoothing the histograms by a kernel density estimation and subsequent fitting of a bimodal propability density function on the data. The mean membrane potentials of the LP fractions at different osmolalities were depolarized to values between -50 and -60 mV, those of the HP fractions represented a sigmoidally shaped curve superposing a hyperpolarization. A computer model of an excitable cell, possessing a volume-dependent NKCC activity, was fitted to the HP data. The hyperpolarization is due to passive cell behaviour, which is cell shrinkage and the resulting increase of the intracellular K⁺ concentration. The dominant depolarizing sigmoidal distortion of the response curve is caused by the shrinkage-induced stimulation of the NKCC activity, which could be blocked by bumetanide (10 mM). In rat diaphragm NKCC is activated to its maximum at an extracellular osmolality of 340 mOsmol/kg, in rat FDB the maximum occurs at an osmolality of 320 mOsmol/kg.

Conclusions: 

The apparent linear relationship between osmolality and membrane depolarization caused by NKCC activation is based on the calculation of means under the assumption of a unimodal distribution. Actually, there is a bimodal distribution due to the bistability of the membrane potential [2]. Our analysis confirmed the hypothesis for a sigmoidal NKCC activation curve in the well polarized (HP) fraction of fibers. Additionally, we revealed a smaller linear hyperpolarization based on cell shrinkage.

[1] Ferenczi EA, et al., J Physiol. 2004, 555: 423–38; [2] Geukes Foppen RJ et al., Comp Biochem Physiol A Mol Integr Physiol. 2001, 130: 533–8