Presynaptic calcium channels in rat cortical synaptosomes: fast- kinetics of phasic calcium influx, channel inactivation, and relationship to nitrendipine receptors

Abstract

Fast-mixing and rapid-filtration techniques were used to analyze the kinetics of potassium-depolarization-dependent (delta K+ = 47.5 mM) influx of 45Ca into synaptosomes, in the time range from 50 msec to 5 sec. The results are consistent with the presence in synaptosomes of a homogeneous population of voltage-sensitive Ca channels. With 1 mM Cao in the medium, the delta K+-dependent Ca influx has a single-exponential time course with the half-life, t1/2 approximately 0.5-0.7 sec. Ca influx, measured between 0.1 and 10 mM Cao, shows half-saturation (KCa) at 1.5 mM Cao and has the limiting value (JCamax) of 5.9 nmol/sec/mg protein, or a current of approximately 0.06 pA/micron2 surface area. The estimated density of functional Ca channels is 0.6-6 micron-2. Voltage- and time-dependent inactivation of Ca channels was measured in synaptosomes predepolarized in 52.5 mM Ko+ with Ca omitted from the medium. Channel inactivation is a single-exponential process with a half-life of t1/2 approximately 2.3 sec. Channel recovery in 5 mM Ko+ media is likewise a single-exponential process with a half-life of t1/2 approximately 4.3 sec. The slower rate of voltage-dependent channel inactivation than of decay of Ca influx suggests that Ca entry into synaptosomes terminates by a mechanism that depends on Ca influx itself. Synaptosomes contain 200 fmol/mg protein, or approximately 6 micron-2 high-affinity (KD = 0.12 nM) 3H-nitrendipine binding sites; however, nitrendipine at concentrations greater than 10(4) X KD is without effect on the phasic influx of Ca measured at 215 msec with either 1.0 or 0.1 mM Cao. This suggests that Ca channels characterized in this study belong to a class of dihydropyridine-insensitive channels.

Rise of Intrasynaptosomal Ca2+ Level and Activation of Nitric Oxide Synthase in Adult Rat Cerebral Cortex Pretreated with 3-5-3′-L-Triiodothyronine

“…However, T 3 has no such effects on nondepolarized synaptosomal suspension ( Figure 1A, Figure 1B). The maximal rise in [Ca 2ϩ ] i within 5 s of T 3 application in depolarized synaptosomes indicates an early event of Ca 2ϩ accumulation that may be mediated through the altered activity of the voltage-gated Ca 2ϩ channel (Nachshen and Blaustein 1980;Leslie et al 1983;Nachshen 1985;Suszkiw et al 1986;McMahon and Nicholls 1991;Sihra et al 1992;Turner et al 1993). A dose-dependent rise in T 3 -induced [Ca 2ϩ ] i is evident between 0.1 nM and 1.0 nM, 1.0 nM and 10 nM, 10nM and 100 nM doses, having a maximum significant rise at 100 nM dose.…”

Characterization of a calcium current in a vertebrate cholinergic presynaptic nerve terminal

“…They are, however, consistent with previous studies on presynaptic nerve terminal Ca channels at many fast-transmitting synapses using more indirect techniques. These include insensitivity to DHP blockers (see Nachsen and Blaustein, 1979;Pemey et al, 1986;Miller, 1987;Himing et al, 1988;Suszkiw et al, 1989), slow inactivation (Suszkiw et al, 1986), and sensitivity to w-CTX (Kerr and Yoshikami, 1984;Yoshikami et al, 1989).…”

The glutamatergic nerve terminal

“…'Ta entry in response to KCI depolarization is strongly biphasic: an initial rapid entry of 45Ca2+ declines within 1 s to a slow uptake which is retained for the duration of the depolarization (Nachshen and Blaustein, 1979, 1982Scott et al, 1980;Suszkiw and O'Leary, 1983;Coutinho et al, 1984;Turner and Goldin, 1985;Nachshen, 1985b;Suszkiw et al, 1986). Only this initial phase can be determined with any accuracy and it is here that the first evidence for a heterogeneity of presynaptic Ca" channels becomes apparent.…”

“…The marine hunting snails Conus geographus and Conus magus produce a variety of peptide Ca2+ channel antagonists, two of which, o-conotoxin GVIA (Cruz and Olivera, 1986) and w-conotoxin GVIIC (Hillyard et al, 1992), show distinctive patterns of inhibition of presynaptic Ca2+ channels. w-Conotoxin-GVIA is much more effective in inhibiting KC1-evoked 45Ca entry into chick or frog brain synaptosomes than into mammalian synaptosomes (Suszkiw et al, 1987;Lundy et a]., 1989;Scheer, 1990), while o-conotoxin-GVIIC is effective against rat brain synaptosomes (Hillyard et al, 1992). Both toxins bind to crude brain membranes although apparently to distinct sites (Hillyard et al, 1992).…”

“…Although presynaptic terminals possess high-affinity binding sites for dihydropyridine antagonists of L-type Ca2+ channels (Turner and Goldin, 1985;Suszkiw et al, 1986;Carvalho et al, 1986;Dunn, 1988;Massieu and Tapia, 1988) the consensus is that Ca" fluxes and transmitter release are insensitive to the inhibitors (Nachshen and Blaustein, 1979 ;Daniell and Leslie, 1983;Suszkiw et al, 1986Suszkiw et al, , 1989Reynolds et al, 1986;Carvalho et al, 1986;Massieu and Tapia, 1988) although a partial dihydropyridine sensitivity has also been reported for the initial rapid phase of 45Ca uptake (Turner and Goldin, 1985;Woodward et al, 1988a).…”