MULTIPLE LEVELS OF NATIVE CARDIAC NA+ CHANNELS AT ELEVATED-TEMPERATURES MEASURED WITH HIGH-BANDWIDTH LOW-NOISE PATCH CLAMP

Currents through single Na+ channels were studied in cell-attached patches of enzymatically dispersed heart cells of the mouse with a low-noise patch-clamp technique that allows evaluation of current levels at temperatures of up to 35-degrees-C with bandwidths of up to 13 kHz. Noise arising from the pipette and the holder was reduced by the use of short (total length 8 mm) patch pipettes, which were sealed at their end with oil and inserted for only 1.5 mm into an appropriately tipped holder. At 9-degrees-C (filter 5 kHz), channel openings were regularly dominated by one open level, and amplitude histograms could be fitted with high accuracy with a sum of Gaussian curves. Above 24-degrees-C (filter 10 or 13 kHz), however, channel-open levels were heterogeneous with maximum levels of up to 4.5 pA at -50 mV. Amplitude histograms with improved resolution, based on variance calculation with window widths of 75 mus or 195 mus, confirmed the observed heterogeneity of open levels. Regular level patterns were not found. The frequency of the largest levels strongly varied from patch to patch and intermediate levels were always the most frequent. A corresponding dissociation of amplitudes was also observed at 35-degrees-C. Averaged currents, formed from trace ensembles including only levels below arbitrarily set borders, obeyed equal kinetics. It is concluded that at low temperature the conductance of single Na+ channel currents is much more homogeneous than at 24-degrees-C and above, where the same channels have multiple open states with different conductance.