ACTIVATION OF SMOOTH MUSCLE BK CHANNELS BY HYDROCHLOROTIAZIDE REQUIRES CELL INTEGRITY AND PRESENCE OF BK BETA-1 SUBUNITS.

ORSI, F; FLOREZ CASTRO, A; ASUAJE, A; MILESI, V; MARTÍN, P

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Hydrochlorothiazide (HCTZ) is used to treat hypertension, their efficacy is linked to a chronic vasodilatory effect. Previous studies suggest that activation of the large conductance voltage- and Ca2+-dependent K+ (BK) channel is responsible for HCTZ-induced vasodilatory effect. However, direct electrophysiological evidence supporting this claim is lacking. BK channels can be associated with accessory β-subunits, which confer specific biophysical and pharmacological characteristics. The β1-subunit is mainly expressed in smooth muscle cells (SMCs). Methods: We evaluated the effect of HCTZ on BK channel activity using patch-clamp technique on SMCs from human umbilical artery (HUASMCs) and in HEK293T cells expressing the BK channel. Paired t-tests were used to compare two groups. Results: Using the whole-cell configuration (WCC) in HEK cells expressing the BK channel with β1-subunit we observed that HCTZ raised the current amplitude with an EC50 of 28.4 µM (pD2=4.5 ± 0.2, n:8). However, 30 µM HCTZ did not change the channel activity when it was evaluated in the same cells in the inside-out configuration (IOC), where cell integrity is lost (%current increase (+90 mV): 12.8 ± 38.1, P>0.05; n:4), suggesting an indirect action. Then, we tested the effect of HCTZ in WCC currents in HEK cells expressing the BK channel without any β-subunit. In this condition, 100 µM HCTZ did not change the BK activity (%current increase (+40 mV): 13.9 ± 15.7, P>0.05; n:4). These experimental results were repeated in HUASMCs. Consistent with the previous results, 10 µM HCTZ caused significant activation of BK current in WCC (528 ± 215 to 1379 ± 132 pA at +40mV, n: 4, p 0.05). Conclusion: A β1-subunit-dependent mechanism that requires SMC integrity leads to HCTZ-induced BK channel activation.