CONICET | Buscador de Institutos y Recursos Humanos

Human vascular smooth muscle cells (SMC) exposed to extracellular ATP (ATPe) evoked a transient inward ionic current, carried by Na+ and Ca2+, characterized by a fast activation followed by a peak current decrease. Among the different roles this Ca2+ influx could have on the SMCs, we focus on its possible functional coupling with the large conductance voltage and Ca2+ activated K+ channels (BKCa). Since this K+ channel has a high Ca2+ sensitivity, it could be activated by the ATP-induced-calcium- influx, producing hyperpolarization of SMC and subsequently modulate the contractile state of these cells. Whole-cell patch clamp experiments were performed applying consecutives voltage protocols designed by a 0.5 s voltage step from -50 to +80 mV (which evokes a BKCa current), followed by a 2 s voltage ramp from +80 to -80 mV. ATPe was added at voltage values below 0 mV, where the impulse force is favorable to produce an inward cationic current through P2X channels. Immediately after 100 μM ATPe addition we observed a significant increase in BKCa current and a significant left shift of the ramp evoked current(n=6, p<0.05, paired Student?s test). To exclude a direct activation of BKCa channel by ATP, we worked in cell attached patch clamp configuration, where the membrane being under recording it is not in contact with ATPe and cell integrity is maintained. We observed BKCa channel activation, translated as a significant higher open probability value (NPo), after application of 100uM ATPe (control NPo: 0.0197 ± 0.0128; after ATPeNPo: 0.0552 ± 0.0239 at 0 mV, n=6, p<0.05, paired Student?s t test). We also tested100 μM ATPe in a Ca2+ free extracellular solution observing no effect on BKCa channel activity, which suggests that Ca2+ influx through P2X receptors is essential to activate the K+ channel.

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