Ionic currents in single smooth muscle cells of the canine renal artery

Abstract

Membrane currents from single smooth muscle cells enzymatically isolated from canine renal artery were recorded using the patch-clamp technique in the whole-cell and cell-attached configurations. These cells exhibited a mean resting potential, input resistance, membrane time constant, and cell capacitance of -51.8±2.1 mV, 5.2±0.98 GΩ, 116.2±16.4 msec, and 29.1±2.0 pF, respectively. Inward current, when elicited from a holding potential of -80 mV, activated near -50 mV, reached a maximum near 0 mV and was sensitive to the dihydropyridine agonist Bay K 8644 and dihydropyridine antagonist nisoldipine. Two components of macroscopic outward current were identified from voltage-step and ramp depolarizations. The predominant charge carrier of the net outward current was identified as K+ by tail-current experiments (reversal potential, -61.0±0.8 mV in 10.8 mM [K+]o/140 mM [K+]i). The first component was a small, low-noise, voltage- and time-dependent current that activated between -40 and -30 mV (IK(dr)), and the second component was a larger, noisier, voltage- and time-dependent current that activated at potentials positive to +10 mV (IK(Ca)). Both IK(dr) and IK(Ca) displayed little inactivation during long (4-second) voltage steps. IK(ca) and IK(dr) could be pharmacologically separated by using various Ca2+ and K+ channel blockers. IK(Ca) was substantially inhibited by external NiCl2 (500 μM), CdCl2 (300 μM), EGTA (5 mM), tetraethylammonium (Ki at +60 mV, 307 μM), and charybdotoxin (100 nM) but was insensitive to 4-aminopyridine (0.1-10 mM). IK(dr) was inhibited by 4-aminopyridine (Ki at +10 mV, 723 μM) and tetraethylammonium (Ki at +10 mV, 908 μM) but was insensitive to external NiCl2 (500 μM), CdCl2 (300 μM), EGTA (5 mM), and charybdotoxin (100 nM). Two types of single K+ channels were identified in cell-attached patches. The most abundant K+ channel that was recorded exhibited voltage-dependent activation, was blocked by external tetraethylammonium (250 μM), and had a large single-channel conductance (232±12 pS with 150 mM K+ in the patch pipette, 130±17 pS with 5.4 mM K+ in the patch pipette). The second channel was also voltage dependent, was blocked by 4-aminopyridine (5 mM), and exhibited a smaller single-channel conductance (104±8 pS with 150 mM K+ in the patch pipette, 57±6 pS with 5.4 mM K+ in the patch pipette). These results suggest that depolarization of canine renal artery cells opens dihydropyridine-sensitive Ca2+ channels and at least two K+ channels. The two time-dependent K+ currents (IK(Ca) and IK(dr)) reflect the behavior of two distinct K+ channels. According to the pharmacology of the whole-cell and single-channel experiments, IK(Ca) is primarily carried by the large conductance Ca2+-activated K+ channel, and IK(dr) is carried by the smaller conductance delayed rectifier K+ channel.