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J Physiol. 1993 Dec;472:33-44.

Microheterogeneity of subsarcolemmal sodium gradients. Electron probe microanalysis in guinea-pig ventricular myocytes.

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Department of Physiology, University of Tübingen, Germany.


1. The effect of stimulation on possible subsarcolemmal sodium accumulation was studied in ventricular myocytes (2 mM [Ca2+]o, 36 degrees C). By trains of eighteen paired voltage-clamp pulses (180 ms to 0 mV, 20 ms to -45 mV, 180 ms to +50 mV, 620 ms to -45 mV) unloaded contractions were potentiated to an optimum. 2. Potentiation reversibly enlarged and prolonged the diastolic tail currents due to Na(+)-Ca2+ exchange. Eighteen pulse pairs were estimated to provide a sodium influx that could increment the total intracellular sodium concentration (sigma Na(i)) by no more than 0.5 mM. 3. Potentiation reversibly increased the current at +50 mV and made it more noisy. Cell-attached recordings with a second electrode attributed this noise to the activation of K+ (Na) channels. In inside-out patches, a comparable channel activity was obtained with 40 mM sodium. Hence, the cell-attached recordings suggest that potentiation can increase intracellular sodium concentration to 40 mM. 4. Electron probe microanalysis (EPMA) measured sigma Na in a volume within 20 nm of the inner side of the sarcolemma. Potentiation reversibly increased sigma Na20nm to 40 +/- 7 mM. When stimulation was terminated, sigma Na20nm fell within 8 s to 37 +/- 8 mM and within 3 min to 19 +/- 6 mM. In unstimulated cells sigma Na20nm was 17 +/- 5 mM. 5. In potentiated cells, shock-frozen at early systole, sigma Na fell with a space constant of 28 nm from the sarcolemma to the centre; at 1 microns distance sigma Na was 12 +/- 3 mM. The steep gradient suggests that sodium does not freely diffuse and sigma Na20nm is controlled by transmembrane fluxes rather than by cell dialysis. 6. sigma Na20nm data were distributed with peaks at 5, 30 and 60 mM. Quantitative elemental digital imaging demonstrated patches with 60-80 mM sigma Na20nm alternating with others of 0-15 mM sigma Na20nm. This 'sodium microheterogeneity' suggests that Ca2+ efflux at low sigma Na20nm and K+(Na) channel activation at high sigma Na20nm can operate simultaneously.

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