Functional coupling between BKCa and P2X channels in human vascular smooth muscle cells

ENRIQUE, N; ASUAJE, A; MONCADA, M; MARTÍN, P; MILESI, V

Rio de Janeiro

Congreso; 38th IUPS Congress (International Union of Physiology Sciences); 2017

International Union of Physiology Sciences

Human vascular smooth muscle cells (SMC) exposedto extracellular ATP (ATPe) evoked atransient inward ionic current, carried by Na+ and Ca2+, characterizedby a fast activation followed by a peak current decrease toward the baselinevalue,  typical of desensitization processof P2X purinergic receptors. Among the different roles this Ca2+influx could have on the SMCs physiology,we focus on its possible functional coupling with the large conductance voltageand Ca2+ activated K+ channels (BKCa). Since this K+channel has a high Ca2+ sensitivity, it could be activated by theATP-induced-Ca2+-influx, producing hyperpolarization of SMC andsubsequently modulate the contractile state of these cells. Whole-cell patch clamp experiments wereperformed applying two consecutive voltage protocols designed by a 0.5 svoltage step from -50 to +80 mV (which evokes a BKCa current), followed by a 2s voltage ramp from +80 to -80 mV. ATPe wasadded at voltage values below 0 mV, where the impulse force is favorable toproduce an inward cationic current through P2X channels. Immediately after 100 µM ATPe addition we observed a significant increase in BKCa current and asignificant left shift of the ramp evoked current (n=6, p<0.05, pairedStudent?s t test), suggesting that BKCa current increases as a consequence of ATPe addition.To exclude a direct activation of BKCa channelby ATP, we worked in cell attached patch clamp configuration, where themembrane being under recording it is not incontact with ATPe and cell integrity ismaintained. We observed BKCa channel activation, translated as a significanthigher open probability value, after application of 100 uM 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).Moreover, after ATPe wash out, a second stimulus with ATPe did not have any effect on BKCa activity, reflectingthe expected behavior for a desensitized P2X channel.We also tested 100 µM ATPe in a Ca2+ free extracellularsolution observing no effect on BKCa channel activity, which suggests that Ca2+influx through P2X receptors is essential to activate the K+channel.These results show that extracellular ATP,acting as an autocrine or paracrine signal,could play an important role as a regulatory mechanism of the vascularresistance involving BKCa channel as a new functional partner.