Abstract
BACKGROUND AND PURPOSE: In arterial smooth muscle cells (myocytes), intravascular pressure stimulates membrane depolarization and vasoconstriction (the myogenic response). Ion channels proposed to mediate pressure-induced depolarization include several transient receptor potential (TRP) channels, including TRPM4, and transmembrane protein 16A (TMEM16A), a Ca(2+) -activated Cl(-) channel (CaCC). 9-Phenanthrol, a putative selective TRPM4 channel inhibitor, abolishes myogenic tone in cerebral arteries, suggesting that either TRPM4 is essential for pressure-induced depolarization, upstream of activation of other ion channels or that 9-phenanthrol is non-selective. Here, we tested the hypothesis that 9-phenanthrol is also a TMEM16A channel blocker, an ion channel for which few inhibitors have been identified. EXPERIMENTAL APPROACH: Patch clamp electrophysiology was used to measure rat cerebral artery myocyte and human recombinant TMEM16A (rTMEM16A) currents or currents generated by recombinant bestrophin-1, another Ca(2+) -activated Cl(-) channel, expressed in HEK293 cells. KEY RESULTS: 9-Phenanthrol blocked myocyte TMEM16A currents activated by either intracellular Ca(2+) or Eact , a TMEM16A channel activator. In contrast, 9-phenanthrol did not alter recombinant bestrophin-1 currents. 9-Phenanthrol reduced arterial myocyte TMEM16A currents with an IC50 of ∼12 μM. Cell-attached patch recordings indicated that 9-phenanthrol reduced single rTMEM16A channel open probability and mean open time, and increased mean closed time without affecting the amplitude. CONCLUSIONS AND IMPLICATIONS: These data identify 9-phenanthrol as a novel TMEM16A channel blocker and provide an explanation for the previous observation that 9-phenanthrol abolishes myogenic tone when both TRPM4 and TMEM16A channels contribute to this response. 9-Phenanthrol may be a promising candidate from which to develop TMEM16A channel-specific inhibitors.