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. 2010 Apr 12;107(17):8005–8010. doi: 10.1073/pnas.0912029107

Fig. 5.

Fig. 5.

Carbachol-induced inhibition of BK channels in tracheal smooth muscle cells (TSMCs) shows the same characteristics as PKC-induced inhibition of BK channels in HEK293 cells. (A–C) Whole-cell outward currents (Iout) were elicited from a holding potential of −10 mV by depolarizing freshly isolated TSMCs every 5 s for 300 ms from −60 to +80 mV in 10-mV increments. Means ± SEM are shown. The original current recordings are at +80 mV, and reflect interventions as indicated by the symbols in the corresponding figure. The pipette solution contained 0.3 μM Ca2+. (A) Carbachol (CCh, 10 μM) decreases Iout and prevents channel regulation by activators of PKA (cAMP = 300 μM Sp-5,6-DCI-cBIMPS) and PKG (cGMP = 300 μM 8-pCPT-cGMP). Current-voltage relationships of 16 cells from 5 mice (Ctr and CCh), of 10 cells from 4 mice (CCh + cGMP), and of 6 cells from 3 mice (CCh + cAMP) are shown. (B) In the absence of CCh, only the activator of PKG (cGMP) enhances Iout. Ctr (19 cells from 9 mice), cAMP (6 cells from 3 mice), Iso (10 μM isoproterenol; 7 cells from 3 mice), and cGMP (6 cells from 3 mice). (C) The PKC inhibitor Ro31-8220 (1 μM Ro) partially inhibits the CCh effect and restores the stimulatory action of the PKG activator (cGMP). Iout of 6 cells from 3 mice is presented at +80 mV. (D) Dephosphorylation of BK channels by exposing inside-out patches from TSMCs to ATP-free solution containing 5 μM of the PKC inhibitor peptide PKC19–31, switches NPo activation from PKG to PKA. Means ± SEM of NPo at +40 mV with n (from 4 mice) in parentheses. (E and F) The phosphatase 1 inhibitor peptide (20 nM PPI2) decreases NPo of inside-out patches from TSMCs via a PKC-dependent pathway and prevents activation by PKG and PKA. Means ± SEM of NPo at +40 mV. The original traces in E show single-channel recordings from a representative patch. (E) 13 patches from 6 mice (Ctr and PPI2), 7 patches from 3 mice (PPI2 + PKG), 6 patches from 3 mice (PPI2 + PKA), and (F) 6 patches from 4 mice. (D–F) The intracellular (bath) Ca2+ concentration was 0.3 μM. **P < 0.01; ***P < 0.001; ns, not significant.