Ca(2+)-activated and voltage-gated K+ currents in smooth muscle cells isolated from human mesenteric arteries.
1. Smooth muscle cells were enzymatically isolated from arteries dissected from mesenteric fat removed from patients undergoing routine surgery. The whole-cell patch clamp technique was used to characterize the potassium (K+) currents and passive electrical properties of these cells, using high-K(+)-containing pipette solutions with either 0.2 mM EGTA or 10 mM EGTA and 10 mM BAPTA. 2. Cell capacitance, which is proportional to membrane surface area, was normally distributed around a value of 46 pF, and independent of artery size between 0.4 and 3.6 mm. The mean membrane potential measured under current clamp was -44.1 +/- 1.9 mV (n = 52). 3. Cells dialysed with 0.2 mM EGTA in order to weakly buffer intracellular Ca2+ demonstrated a noisy outward current with an apparent threshold near -30 mV, upon which were superimposed spontaneous transient outward currents (STOCs). In the presence, but not the absence, of extracellular Ca2+, this current was potentiated if the holding potential was depolarized into the voltage range between -40 and +50 mV. This potentiation had a bell-shaped potential dependency which reflected the activation of voltage-gated Ca2+ channels in these cells. 4. The noisy current was blocked by externally applied tetraethylammonium (the dissociation constant, Kd = 0.85 mM), as were STOCs. This current was also reduced by about 40% by 8 nM charybdotoxin, and was transiently potentiated by 10 mM caffeine. The characteristics of this current therefore suggested that it was carried by large-conductance Ca(2+)-activated K+ channels. 5. Dialysis of human mesenteric arterial cells with 10 mM EGTA and 10 mM BATPA was not able to completely suppress the Ca(2+)-activated current, and reduced by approximately 50% the amplitude of the outward current recorded at positive potentials. 6. Depolarization of strongly Ca(2+)-buffered cells in the presence of 30 mM TEA to block Ca(2+)-activated K+ channels revealed a residual outward current which had both transient and sustained components. These were blocked by 4-aminopyridine (4-AP) with a similar efficiency (Kd was 1.04 and 1.16 mM at +60 mV for transient and sustained current, respectively), but the voltage ranges over which they inactivated, and their rates of recovery from inactivation, were significantly different. 7. The transient and sustained currents had different sensitivities to external Ca2+ and Cd2+ ions. Ca2+ (5 mM) significantly reduced the amplitude and shifted the voltage dependency of inactivation of the transient but not the sustained component of the outward current. Cd2+ (0.2 mM) reduced the transient current by about 30% without affecting the sustained component amplitude.(ABSTRACT TRUNCATED AT 400 WORDS)
Smirnov
S V
SV
Department of Pharmacology, United Medical School, Guy's Hospital, London.
Aaronson
P I
PI
D002104:Cadmium / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D018999:Charybdotoxin; D004553:Electric Conductivity; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D008638:Mesenteric Arteries / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D012604:Scorpion Venoms / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D012604:Scorpion Venoms; D013757:Tetraethylammonium Compounds; D002104:Cadmium; D018999:Charybdotoxin; D002110:Caffeine; D019789:Tetraethylammonium; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium