Ca2+ -activated Cl- channels (TMEM16A) underlie spontaneous electrical activity in isolated mouse corpus cavernosum smooth muscle cells

Abstract

Penile detumescence is maintained by tonic contraction of corpus cavernosum smooth muscle cells (CCSMC), but the underlying mechanisms have not been fully elucidated. The purpose of this study was to characterize the mechanisms underlying activation of TMEM16A Ca²⁺ -activated Cl^- channels in freshly isolated murine CCSMC. Male C57BL/6 mice aged 10-18 weeks were euthanized via intraperitoneal injection of sodium pentobarbital (100 mg.kg^-1 ). Whole-cell patch clamp, pharmacological, and immunocytochemical experiments were performed on isolated CCSM. Tension measurements were performed in whole tissue. TMEM16A expression in murine corpus cavernosum was confirmed using immunocytochemistry. Isolated CCSMC developed spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarizations (STDs) in current clamp mode of the whole cell, perforated patch clamp technique. STICs reversed close to the predicted Cl^- equilibrium potential and both STICs and STDs were blocked by the TMEM16A channel blockers, Ani9 and CaCC(inh)-A01. These events were also blocked by GSK7975A (ORAI inhibitor), cyclopiazonic acid (CPA, sarcoplasmic reticulum [SR] Ca^2+- ATPase blocker), tetracaine (RyR blocker), and 2APB (IP₃ R blocker), suggesting that they were dependent on Ca²⁺ release from intracellular Ca²⁺ stores. Nifedipine (L-type Ca²⁺ channel blocker) did not affect STICs, but reduced the duration of STDs. Phenylephrine induced transient depolarizations and transient inward currents which were blocked by Ani9. Similarly, phenylephrine induced phasic contractions of intact corpus cavernosum muscle strips and these events were also inhibited by Ani9. This study suggests that contraction of CCSM is regulated by activation of TMEM16A channels and therefore inhibition of these channels could lead to penile erection.

FIGURE 1

Immunofluorescence detection of TMEM16A in freshly isolated CCSM cells. (ai) representative confocal photomicrograph showing that freshly isolated mouse CCSM cells were immunoreactive to a selective TMEM16A antibody, demonstrated by the strong immunofluorescence signal detected [green] (n = 3 from 3 animals). (aii) bright field image of the same cell as in ai. (bi) negative control, representative confocal photomicrograph showing no immunofluorescence detected in detrusor SM cell exposed to the TMEM16A antibody (n = 3 from 3 animals). (bii) bright field image of the same cell as in bi. (c) Positive control, representative confocal photomicrographs showing robust TMEM16A staining in mouse jejunum tissue strip (n = 3 from 3 animals). (d) Smooth muscle cells were identified by smooth muscle myosin positive immunoreactivity [green] (n = 3 from  3 animals)

FIGURE 2

Two different TMEM16A‐specific blockers inhibited chloride tail currents. (ai) the cell was held at −60 mV and stepped to 0 mV. It was then stepped down to −80 mV to evoke a tail current. Representative traces showing the reversible effect of 1 μM Ani9 on tail current. (aii) summary data showing the effect of 1 μM Ani9 on tail current (n = 6 cells from 6 animals, *p < 0.05; one way ANOVA). (bi) representative traces showing the reversible effect of 3 μM CaCC(inh)‐A01 on tail current. (bii) summary data showing the effect of 3 μM CaCC(inh)‐A01 on tail current (n = 6 cells from 6 animals, *p < 0.05; one way ANOVA)

FIGURE 3

Effect of low Cl⁻ hanks buffer on tail currents. (a) an isolated CCSMC was held at −60 mV and stepped to 0 mV followed by a voltage ramp from −50 to +50 mV. Recordings were made in 135 mM external Cl⁻ (control) and 49 mM external Cl⁻ (low Cl⁻) solutions. Capacitive current in all data presented was eliminated by subtracting from traces evoked in the presence of nifedipine, revealing the difference current. (b) the shift in the reversal potential of the tail current is consistent with the current being carried through Ca²⁺‐activated Cl⁻ channels (n = 6 cells from 6 animals; *p < 0.05; paired t‐test)

FIGURE 4

Two different TMEM16A‐specific blockers abolished STICs and STDs. (a) an example showing the effect of 1 μM Ani9 and 3 μM CaCC(inh)‐A01 on STICs held under voltage clamp at −60 mV. (bi) summary of the effect of 1 μM Ani9 on STIC amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (bii) summary of the effect of 1 μM Ani9 on STIC frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (biii) summary of the effect of 3 μM CaCC(inh)‐A01 on STIC amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (biv) summary of the effect of 3 μM CaCC(inh)‐A01 on STIC frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (c) an example showing the effect of 1 μM Ani9 and 3 μM CaCC(inh)‐A01 on STDs in current clamp mode. (di) summary of the effect of 1 μM Ani9 on STD amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (dii) summary of the effect of 1 μM Ani9 on STD frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (diii) summary of the effect of 3 μM CaCC(inh)‐A01 on STD amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (div) summary of the effect of 3 μM CaCC(inh)‐A01 on STD frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA)

FIGURE 5

Involvement of CRAC channels and Ca²⁺ stores in activation of STICs and STDs. (a) an example showing the effect of 3 μM GSK7975A and 10 μM CPA on STICs held under voltage clamp at −60 mV. (bi) summary of the effect of 3 μM GSK7975A on STICs amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (bii) summary of the effect of 3 μM GSK7975A on STIC frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (biii) summary of the effect of 10 μM CPA on STICs amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (Biv) summary of the effect of 10 μM CPA on STIC frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (c) an example showing the effect of 3 μM GSK7975A and 10 μM CPA on STDs in current clamp mode. (di) summary of the effect of 3 μM GSK7975A on STD amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (dii) summary of the effect of 3 μM GSK7975A on STD frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (diii) summary of the effect of 10 μM CPA on STD amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (div) summary of the effect of 10 μM CPA on STD frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA)

FIGURE 6

Involvement of both RyR and IP₃R in activation of STICs and STDs. (a) an example showing the effect of 100 μM tetracaine and 100 μM 2APB on STICs held under voltage clamp at −60 mV. (bi) summary of the effect of 100 μM tetracaine on STIC amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (bii) summary of the effect of 100 μM tetracaine on STIC amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (biii) summary of the effect of 100 μM 2APB on STIC frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (biv) summary of the effect of 100 μM 2APB on STIC amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (c) an example showing the effect of 100 μM tetracaine and 100 μM 2APB on STDs in current clamp mode. (di) summary of the effect of 100 μM tetracaine on STD amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (dii) summary of the effect of 100 μM tetracaine on STD frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (diii) summary of the effect of 100 μM 2APB on STD amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (div) summary of the effect of 100 μM 2APB on STD frequency (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA)

FIGURE 7

Involvement of L‐type Ca²⁺ channel in activation of STICs and STDs. (a) an example showing the effect of 1 μM nifedipine on STICs held under voltage clamp at −60 mV. (bi) summary of the effect of 1 μM nifedipine on STIC amplitude (n = 6 cells from 6 animals; *p > 0.05; one way ANOVA). (bii) summary of the effect of 1 μM nifedipine on STIC frequency (n = 6 cells from 6 animals; *p > 0.05; one way ANOVA). (c) an example showing the effect of 1 μM nifedipine on STDs in current clamp mode. (di) summary of the effect of 1 μM nifedipine on STD amplitude (n = 6 cells from 6 animals; *p > 0.05; one way ANOVA). (dii) summary of the effect of 1 μM nifedipine on STD frequency (n = 6 cells from 6 animals; *p > 0.05; one way ANOVA)

FIGURE 8

Involvement of L‐type Ca²⁺ channel in the plateau phase of STD. (a) 1 μM nifedipine reduced the duration of STDs in CCSM. (b) Summary data showing the effect of 1 μM nifedipine on the mean duration of STDs (n = 6 cells from 6 animals; *p < 0.05; paired t‐test)

FIGURE 9

Ani9 inhibited phenylephrine‐induced transient inward currents and transient depolarizations. (a) 300 nM phenylephrine evoked a large, transient inward current, followed by STICs which were susceptible to blockade by 1 μM Ani9. There was only partial return of phenylephrine response after washout from Ani9 (bi) summary data showing the effect of Ani9 on the amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA) and (bii) frequency of phenylephrine induced depolarizations (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA). (c) 300 nM phenylephrine evoked regular, oscillating depolarizations, which were susceptible to block by 1 μM Ani9. (di) summary data showing the effect of Ani9 on the amplitude (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA) and (dii) frequency of phenylephrine induced depolarizations (n = 6 cells from 6 animals; *p < 0.05; one way ANOVA)

FIGURE 10

Ani9 reduced the frequency of phenylephrine‐induced contractions in CCSM tissue strips. (a) Representative trace showing phasic, oscillatory contractions evoked by 300 nM phenylephrine that are susceptible to blockade by 10 μM Ani9. (bi) summary data showing the effect of 10 μM Ani9 on mean evoked contraction amplitude (n = 6 strips from 6 animals; *p > 0.05; paired t‐test) and (Bii) evoked contraction events per hour (n = 6 strips from 6 animals; *p < 0.05; paired t‐test)