9-Phenanthrol inhibits recombinant and arterial myocyte TMEM16A channels

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.

Figure 1

9-Phenanthrol inhibits TMEM16A currents in cerebral artery smooth muscle cells. (A) Examples of whole-cell TMEM16A currents recorded in the absence (control) and presence of 9-phenanthrol (10 μM) in a smooth muscle cell. (B) Mean grouped data for whole-cell currents: control, n = 8; 9-phenanthrol: 5 μM, n = 7; 10 μM, n = 6; 15 μM, n = 5; 20 μM, n = 4. P was < 0.05 when compared with control for: 5 μM at +80, +100, and +120 mV, 10 μM at −80, −60, +60, +80, +100 and +120 mV; 15 μM at −80, −60, −40, +60, +80, +100 and +120 mV; 20 μM at −80, −60, −40, +40, +60, +80, +100 and +120 mV. (C) Mean data illustrating concentration- and voltage-dependence of 9-phenanthrol inhibition determined from tail currents. (D) Concentration-response mediated inhibition of whole-cell TMEM16A currents by 9-phenanthrol at −80 and +120 mV determined from tail currents (same ns as in B, including 0.1 μM, n = 5; 40 μM, n = 4). (E) Mean data displaying current density generated by a 360 ms depolarizations from −40 to +60 mV every 15 s illustrating the time course of 9-phenanthrol (10 μM) inhibition (n = 8) and washout (n = 5). Data points during solution exchange are not shown due to electrical noise in the recordings. *Indicates P < 0.05.

Figure 2

TMEM16A currents activated by E_act, a TMEM16A channel activator, are blocked by 9-phenanthrol in arterial smooth muscle cells. (A) Examples of Cl⁻ current regulation by E_act and 9-phenanthrol applied in the presence of E_act. E_act (10 μM) and E_act + 9-phenanthrol (10 μM) recordings are from the same cell. (B) Mean data illustrating concentration-dependent inhibition of E_act-activated TMEM16A currents by 9-phenanthrol. P < 0.05 when compared with control for: 100 nM at −90, +70, +90 and +110 mV, 1 μM at −90, −70, −50, +70, +90 and +110 mV; 10 μM at −90, −70, −50, +30, +50, +70, +90 and +110 mV. Control, n = 8; E_act (10 μM), n = 7; E_act + 100 nM 9-phenanthrol, n = 7; E_act + 1 μM 9-phenanthrol, n = 6; E_act + 10 μM 9-phenanthrol, n = 6. (C) Mean data displaying current density generated by 360 ms voltage steps from +10 to +70 mV every 15 s illustrating the time course of 9-phenanthrol inhibition and washout. Data points during exchange are not shown due to electrical noise in the recordings.

Figure 3

Recombinant TMEM16A currents in HEK 293 cells are inhibited by 9-phenanthrol. (A) Original recordings of rTMEM16A currents and inhibition by 9-phenanthrol in HEK293 cells. (B) Mean data for whole-cell currents: control, n = 17; 100 nM 9-phenanthrol, n = 15; 1 μM 9-phenanthrol, n = 14; 10 μM 9-phenanthrol, n = 11; 100 μM 9-phenanthrol, n = 11. (C) Mean data illustrating concentration- and voltage-dependence of 9-phenanthrol rTMEM16A current inhibition determined from tail currents. (D) Concentration-dependent inhibition of rTMEM16A currents at −100 and +100 mV (same ns as in B, including 0.1 μM, n = 15; 300 μM, n = 7). (E) Mean data displaying current density generated by repetitive steps from 0 to +60 mV illustrating 9-phenanthrol inhibition (n = 6) and washout (n = 5). Data points during solution exchange are not shown due to electrical noise in the recordings. *Indicates P < 0.05 when compared with control currents.

Figure 4

9-Phenanthrol does not modulate recombinant bestrophin-1 currents in HEK 293 cells. (A) Representative recordings illustrating that bestrophin-1 expression generates whole-cell Cl⁻ currents in HEK293 cells, and that these currents are unaffected by application of 9-phenanthrol (100 μM). (B) Mean data: control, n = 10. 9-Phenanthrol: 100 nM, n = 10; 1 μM, n = 10; 10 μM, n = 6; 100 μM, n = 5; mock, n = 5.

Figure 5

9-Phenanthrol reduces single recombinant TMEM16A channel open probability and open time and elevates mean closed time in HEK293 cells. (A) Original recordings of mock HEK293 cells and single recombinant TMEM16A channels and inhibition by 9-phenanthrol at −80 mV. Traces were filtered at 2 kHz. Single channel amplitude (B), amplitude histograms for control (C) and 9-phenanthrol (D), open probability (P_o) (E), open time (F) and closed time (G) for control (n = 11) and in the presence of 9-phenanthrol (n = 7). *Indicates P < 0.05.