A NOVEL LARGE-CONDUCTANCE CA-2+-ACTIVATED POTASSIUM CHANNEL AND CURRENT IN NERVE-TERMINALS OF THE RAT NEUROHYPOPHYSIS

1. Nerve terminals of the rat posterior pituitary were acutely dissociated and identified using a combination of morphological and immunohistochemical techniques. Terminal membrane currents were studied using the 'whole-cell' patch clamp technique and channels were studied using inside-out and outside-out patches. 2. In physiological solutions, but with 7 mm 4-aminopyridine (4-AP), depolarizing voltage clamp steps from different holding potentials (-90 or -50 mV) elicited a fast, inward current followed by a slow, sustained, outward current. This outward current did not appear to show any steady-state inactivation. 3. The threshold for activation of the outward current was -30 mV and the current-voltage relation was 'bell-shaped'. The amplitude increased with increasingly depolarized potential steps. The outward current reversal potential was measured using tail current analysis and was consistent with that of a potassium current. 4. The sustained potassium current was determined to be dependent on the concentration of intracellular calcium. Extracellular Cd2+ (80 mum), a calcium channel blocker, also reversibly abolished the outward current. 5. The current was delayed in onset and was sustained over the length of a 150 ms-duration depolarizing pulse. The outward current reached a peak plateau and then decayed slowly. The decay was fitted by a single exponential with a time constant of 9.0 +/- 2.2 s. The decay constants did not show a dependence on voltage but rather on intracellular Ca2+. The time course of recovery from this decay was complex with full recovery taking > 190 s. 6. 4-AP (7 mm), dendrotoxin (100 nm), apamin (40-80 nM), and charybdotoxin (10-100 nm) had no effect on the sustained outward current. In contrast Ba2+ (200 muM) and tetraethylammonium inhibited the current, the latter in a dose-dependent manner (apparent concentration giving 50 % of maximal inhibition (IC50) = 0.51 mM). 7. The neurohypophysial terminal outward current recorded here corresponds most closely to a Ca2+-activated K+ current (I(K(Ca))) and not to a delayed rectifier or I(A)-like current. It also has properties different from that of the Ca2+-dependent outward current described in the magnocellular neuronal cell bodies of the hypothalamus. 8. A large conductance channel is often observed in isolated rat neurohypophysial nerve terminals. The channel had a unit conductance of 231 pS in symmetrical 150 mm K+. 9. With different potassium gradients across the patch of 150:5 and 5:130 (mm), the conductance decreased to 140 and 121 pS, respectively, and the shift in apparent reversal potentials (+42 and -25 mV) were consistent with that of a potassium-selective channel. 10. The open probability (P(o)) of the channel was shown to be dependent on the concentration of calcium (Ca2+) at the intracellular side of the patch. The channel was activated at Ca 2+ concentrations greater than 0.5 mum, and reached apparent full activation at around 5 mum with a half-maximal effective concentration (EC50) for Ca2+ = 1.53 mum. 11. Most patches showed a voltage-sensitive increase in open probability (14.3 mV/e-fold change) with increasing depolarizations of the patch. Other patches showed little voltage-dependent properties. The activation curve for the probability of the channel opening against voltage did not show a significant shift with different intracellular calcium concentrations. 12. Kinetic analysis of the channel activity showed that openings could be well fitted by a single exponential, while closures required two exponentials. The open-time constant (tau(o)) was 49-3 ms, and the closed-time constants were 0.2 ms (tau1) and 11.2 ms (tau2). 13. 4-AP (7 mm), apamin (80 nm), and charybdotoxin (100-360 nm) had no effect on the Ca2+activated K+ channel activity, while both extracellular (I mm) and intracellular (10 muM) Ba2+, and extracellular tetraethylammonium (0-5 mm) decreased the channel P(o). 14. The neurohypophysial large conductance channel appears to be similar to a slow, type II, large-conductance, Ca2+-activated K+ channel previously only reconstituted from total brain membranes, and underlies the macroscopic I(K(Ca)). 15. The existence of this current could help explain why maximal peptide release only occurs in response to bursts of electrical activity invading the nerve terminals.