VIDEO ABSTRACT.

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2؉ -activated K ؉ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the ␣-subunit of the large conductance Large conductance Ca 2ϩ -activated K ϩ (BK Ca ) 1 channels are ubiquitously distributed among tissues and are particularly abundant in smooth muscle (1, 2). The activity of BK Ca channels is regulated by membrane potential, intracellular Ca 2ϩ , and phosphorylation (3, 4). Although BK Ca channels are usually not involved in setting resting potential, they play a key role as a negative feedback mechanism to limit depolarization and contraction (5-7). Activation of BK Ca channels is increased by nitric oxide (NO) and atrial natriuretic peptide, which hyperpolarize the membrane and increase the sensitivity of BK Ca channels to Ca 2ϩ (8 -11). Membrane hyperpolarization closes voltage-dependent Ca 2ϩ channels, reduces Ca 2ϩ influx, and leads to a reduction in intracellular Ca 2ϩ concentration and relaxation (1). NO has been reported to stimulate BK Ca channels directly as well as through stimulation of guanylate cyclase and the subsequent increase in cGMP (12-15). In addition, activation of BK Ca channels plays an important role in NO-induced relaxation of smooth muscle (16 -20). cGMP activates cGMP-dependent protein kinase (PKG), which phosphorylates various cytosolic and membrane proteins that regulate smooth muscle tone either directly or indirectly (21,22). Recent studies in native cells suggest that PKG activates BK Ca channels through phosphorylation of the channel (23). These results are supported by biochemical studies of cloned BK Ca channels, which demonstrate PKG-induced phosphorylation of the channel (24).The primary sequence of BK Ca has been determined using molecular cloning techniques in Drosophila (25) and mammals (26 -28). These studies indicate that BK Ca isoforms belong to the voltage-gated K ϩ (K V ) channel superfamily. The primary sequence of the S1-S6 segment of BK Ca channels is homologous to the corresponding regions in K V channels. The long carboxyl terminus is the region of Ca 2ϩ -sensing (29, 30), and cslo-␣ contains a single high affinity phosphorylation site for PKG at Ser-1072 (3). However additional putative PKG phosphorylation sites have been identified in other splice variants (31). Expression of the slo channel in Xenopus oocytes or mammalian cells gives rise to voltage-gated, Ca 2ϩ -sensitive currents with electrophysiological and pharmacological features similar to those of native BK Ca (32-34). However, although many studies of native cells suggest that BK Ca channel activity is also modulated by various protein kinases (35-38), this property has been difficult to reproduce in cloned channels. Two studies in which slo chann...

The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl− currents (IClCa) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, IClCa was evoked immediately upon membrane rupture but then exhibited marked rundown to ∼20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5′-(β,γ-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, IClCa was ∼100% of initial levels. IClCa recorded with AMP-PNP–containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in IClCa was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater “on” rates, and voltage-dependent closing steps (“off” rates). Our model reproduced well the Ca2+ and voltage dependence of IClCa as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl− channel complex influences current generation dramatically through one or more critical voltage-dependent steps.

A B S T R A C T We have studied the effects of the potassium-blocking agent 4-aminopyridine (4-AP) on the acdon potential and membrane currents of the sheep cardiac Purkinje fiber. 4-AP slowed the rate of phase 1 repolarization and shifted the plateau of the action potential to less negative potentials. In the presence of 4-AP, the substitution of sodium methylsulfate or methanesulfonate for the NaCI of Tyrode's solution further slowed the rate of phase 1 repolarization, even though chloride replacement has no effect on the untreated preparation. In voltage clamp experiments, 4-AP rapidly and reversibly reduced the early peak of outward current that is seen when the Purkinje fiber membrane is voltage-clamped to potentials positive to -20 mV. In addition, 4-AP reduced the steady outward current seen at the end of clamp steps positive to -40 inV. 4-AP did not appear to change the slow inward current observed over the range of -60 to -40 mV, nor did it greatly change the current tails that have been used as a measure of the slow inward conductance at more positive potentials. 4-AP did not block the inward rectifying potassium currents, Is1 and IK2. A phasic outward current component that was insensitive to 4-AP was reduced by chloride replacement. We conclude that the early outward current has two components: a chloride-sensitive component plus a 4-AP-sensitive component. Since a portion of the steady-state current was sensitive to 4-AP, the early outward current either does not fully inactivate or 4-AP blocks a component of time-independent background current.

A B S T R A C T The rapid repolarization during phase 1 of the action potential of sheep cardiac Purkinje fibers has been attributed to a time-and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in Tyrode's solution. We have re-examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac Purkinje fibers in normal Tyrode's solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode's solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time-and voltage-dependent chloride current exists in sheep cardiac Purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.