Calcium-activated potassium channels in isolated presynaptic nerve terminals from rat brain.

Abstract

86Rb efflux was examined in isolated presynaptic nerve terminals (synaptosomes) from rat brain to assess K permeability (PK) changes sensitive to alterations in internal Ca activity. Rb effux from 86Rb-loaded synaptosomes into nominally Ca-free physiological saline (PSS) containing 5 mM-K was .apprx. 0.3-0.4%/s. Raising extracellular K concentration ([K]o), to depolarize the synaptosomes, stimulated the 86Rb eflux. Addition of Ca to the 5 mM-K PSS had no effect, but Ca did further stimulate 86Rb efflux into K-rich solutions. The effect of Ca was graded, with apparent half-maximal activation, Ka .simeq. 0.5 mM-Ca. During depolarization, Ca enters the terminals through voltage-regulated Ca channels and the rise in intracelluar Ca concentration opens certain (Ca-activated) K channels. The Ca-dependent stimulation of 86Rb efflux was greatest during the initial seconds of incubation (component CT) and then declined to a much lower rate (component CS). Much of this change in rate could be attributed to inactivation of voltage-regulated Ca channels and reduced entry of Ca. The Ca-dependent increase in 86Rb efflux was completely inhibited by 100 .mu.M-La. In the presence of Ca, but to in its absence, the Ca ionophore A23187 [calcimycin] stimulated 86Rb efflux both in 5 and 100 mM-K PSS. THe effect of 100 mM-K was quantitatively greater, perhaps because of the increased outward driving force on Rb in depolarized synaptosomes. When synaptosomes were suspended in media containing the voltage-sensitive fluorescent dye, DiS-C3-(5) (1,1''-diphenyl-2,2''-thiocarbocyanine), the addition of Ca + A23187 decreased the fluorescence intensity (= synaptosome hyperpolarization) when the media contained 5 mM-K but not 100 mM-K. In the presence of Ca + A23187, PK was increased and the membrane potential moved closer to the K equilibrium potential, EK. Quinine sulfate, a blocker of Ca-activated K channels, reduced the Ca-stimulated 86Rb efflux with high affinity (apparent half-maximal inhibiton, Ki .simeq. 1 .mu.M). Tetraethylammonium chloride, another agent which blocks Ca-activated K channels, was also a relatively potent inhibitor of Ca-stimulated 86Rb efflux (Ki .simeq. 0.2 mM). The K-channel blocker, 4-aminopyridine, partially inhibited Ca-stimulated86Rb efflux at concentrations < 0.5 mM, but stimulated this efflux at concentrations .gtoreq. 1 mM. Ca-activated K channels are present in mammalian presynaptic nerve terminals, and the physiological and pharmacological properties of these channels can be investigated in this preparation.