K. S. Thorneloe and others 70J. Physiol. 541.1

Macroscopic 4-aminopyridine (4-AP)-sensitive, delayed rectifier K+ current of vascular smooth muscle cells is increased during β-adrenoceptor activation with isoproterenol via a signal transduction pathway involving adenylyl cyclase and cAMP-dependent protein kinase (PKA) (Aiello, E. A., M. P. Walsh, and W. C. Cole. Am. J. Physiol. 268 ( Heart Circ. Physiol. 37): H926–H934, 1995.). In this study, we identified the single delayed rectifier K+(KDR) channel(s) of rabbit portal vein myocytes affected by treatment with isoproterenol or the catalytic subunit of PKA. 4-AP-sensitive KDR channels of 15.3 ± 0.6 pS ( n = 5) and 14.8 ± 0.6 pS ( n = 5) conductance, respectively, were observed in inside-out (I-O) and cell-attached (C-A) membrane patches in symmetrical KCl recording conditions. The kinetics of activation (time constant of 10.7 ± 3.02 ms) and inactivation (fast and slow time constants of 0.3 and 2.5 s, respectively) of ensemble currents produced by these channels mimicked those reported for inactivating, 4-AP-sensitive whole cell KDR current of vascular myocytes. Under control conditions, the open probability ( NP o) of KDR channels of C-A membrane patches at −40 mV was 0.014 ± 0.005 ( n = 8). Treatment with 1 μM isoproterenol caused a significant, approximately threefold increase in NP o to 0.041 ± 0.02 ( P < 0.05). KDR channels of I-O patches exhibited rundown after ∼5 min, which was not affected by ATP (5 mM) in the bath solution. Treatment with the purified catalytic subunit of PKA (50 nM; 5 mM ATP) restored KDRchannel activity and caused NP o to increase from 0.011 ± 0.003 to 0.138 ± 0.03 ( P < 0.05; n = 11). These data indicate that small-conductance, 15-pS KDRchannels are responsible for inactivating the macroscopic delayed rectifier K+ current of rabbit portal vein myocytes and that the activity of these channels is enhanced by a signal transduction mechanism involving β-adrenoceptors and phosphorylation by PKA at a membrane potential consistent with that observed in the myocytes in situ.

We investigated the effects of protons and calcium ions on the voltage-dependent gating of the hyperpolarization-activated, nonselective cation channel current, I(h), in rod photoreceptors. I(h) is a cesium-sensitive current responsible for the peak-plateau sag during the rod response to bright light. The voltage dependence of I(h) activation shifted about 5 mV per pH unit, with external acidification producing positive shifts and alkalinization producing negative shifts. Increasing external [Ca(2+)] from 3 to 20 mM resulted in a large (approximately 17 mV) positive shift in I(h) activation. External [Ca(2+)] (20 mM) blocked pH-induced shifts in activation. Cytoplasmic acidification produced by 25 mM sodium acetate led to a negative shift in inactivation (-9 mV) and internal alkalinization produced with 20 mM ammonium chloride resulted in a positive shift (+6 mV). Surface charge binding and screening theory (Gouy-Chapman-Stern) accounted for the observed shifts in I(h) activation, with the best fit achieved when protons and calcium ions were assumed to bind to distinct sites on the membrane. Since light induces changes in the retinal ionic environment, these results permit us to gauge the degree to which rod light responses could be modified via alterations in I(h) activation.