Fig. 5.
Amiloride sensitivity confirms HSC and NSC channel activity following terbutaline and denopamine treatment in T1 cells. A–B: recordings taken from a continuous cell-attached patch-clamp analysis of terbutaline-treated T1 cells before (t < 10 min) and after 25 nM amiloride diffusion to apical membrane (t > 12 min). Strongly depolarizing potentials of −40 and −60 mV (−Vp) restored HSC channel activity. B: 4 independent observations with y-axis = mean open time showing 25 nM amiloride sensitivity of HSCs. 25 nM amiloride decreased mean open time from 714 ± 361 ms to 448 ± 328 ms; P = 0.004. C–D: recordings taken from a continuous cell-attached patch-clamp analysis of denopamine-treated T1 cells before (control) and after treatment with 25 nM amiloride. Arrows in representative trace indicate closed state of channel, with downward deflections from the closed state depicting entry of Na ions. Submicromolar concentrations of amiloride decreases NSC channel activity but fails to completely block gating properties at 0 mV, −40 mV, −60 mV (−Vp) potentials. D: results from 3 independent observations with y-axis = mean open time. 25 nM amiloride decreased mean open time from 4,096 ± 1,037 ms to 842 ± 223 ms; P = 0.034. E–F: recordings obtained from a continuous cell-attached patch-clamp analysis of denopamine-treated T1 cells before (control) and after treatment with 10 μM amiloride. Again, arrows in representative trace indicate closed state of channel and depict complete blockade of NSC activity with high μM amounts of amiloride.