Cholinergic modulation of apical Na⁺ channels in turtle colon: Analysis of CDPC-induced fluctuations

Current fluctuation analysis was used to investigate the properties apical Na⁺ channels during muscarinic inhibition of active Na⁺ absorption. A reversible Na⁺ channel blocker, 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), was used to induce fluctuations in the short-circuit current (I(sc)). Power density spectra of the CDPC-induced fluctuations exhibited a clearly discernible Lorentzian component, characterized by a corner frequency that was linearly related to CDPC concentration between 20 and 100 μM. The on (k'(on)) and off (k(off)) rate coefficients for the CDPC blocking reaction were k'(on) = 11.1 ± 0.8 rad · s^-1 · μM^-1 and k(off) = 744 ± 53 rad/s, and the microscopic inhibition constant was 67 μM (n = 11). CDPC blocking kinetics were not significantly different after inhibition of I(sc) by 5 μM serosal carbachol. Single-channel Na⁺ current (i(Na)) and the density of open and blocked Na⁺ channels (N(ob)) were estimated from the fluctuations induced by 40 μM CDPC. Under control conditions, i(Na) was 0.43 ± 0.05 pA and N(ob) was 251 ± 42 x 10⁶/cm² (n = 10). After exposure to serosal carbachol (2-10 μM) for 60 min, Na⁺ current and N(ob) were reduced by ~ 50%, but i(Na) was not changed significantly. These results indicate that muscarinic inhibition of electrogenic Na⁺ absorption was associated with a reduction in the number of open Na⁺ channels in the apical membrane. They also suggest that this downregulation of transport involved a coordinated decrease in both apical and basolateral membrane conductances.