Longitudinal distribution of Na + and Ca 2+ channels and β-adrenoceptors on the sarcolemmal membrane of frog cardiomyocytes
Abstract
The distribution of L-type Ca2+ and tetrodotoxin-sensitive Na+ channels and of β-adrenergic receptors was examined in frog ventricular myocytes using the whole-cell patch-clamp technique and a double capillary for extracellular microperfusion.
Rod-shaped cells (250–300 μm long) were sealed at both ends to two patch-clamp pipettes and positioned transversally at different positions between the mouths of two micro-capillaries separated by a thin wall. A combination of nifedipine (1 μm) and tetrodotoxin (0.3 μm) (blocking solution) was added to one capillary in order to inhibit macroscopic Ca2+ and Na+ currents (ICa and INa, respectively) in the part of the cell exposed to this capillary.
Moving the cell in 10–20 μm steps from the control capillary to the capillary containing the blocking solution induced step decreases in ICa and INa amplitudes. Complete block of both currents occurred when the entire cell was exposed to the blocking solution.
Each step decrease in current was due to the loss of activity of the functional Ca2+ and Na+ channels present in the slice of sarcolemmal membrane newly exposed to the blocking solution. These step current changes allowed longitudinal mapping of current density for Ca2+ and Na+ channels on the sarcolemmal membrane.
Addition of a submaximal concentration of isoprenaline (10 nm) to the control capillary induced a local increase in ICa which enabled examination of the distribution of functional β-adrenergic receptors as well.
Our results demonstrate that Ca2+ and Na+ channels and β-adrenergic receptors are equally and essentially uniformly distributed on the sarcolemmal membrane of frog ventricular myocytes.
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