Inhibitory Effects of Nonhydrolyzable Guanine Nucleotides on Neurotransmitter Release at the Squid Giant Synapse.

Abstract

We are examining the role of GTP-binding regulatory proteins in the control of neurotransmitter release. To this end, we injected two nonhydrolyzable guanine nucleotides into the giant presynaptic terminal of the most distal synapse of the stellate ganglion of the squid, Loligo pealei, and studied their effects on transmitter release evoked by action potentials. As an assay of release, the maximum rate of rise of postsynaptic potentials (PSPs) produced by intracellular stimulation of the presynaptic neuron was measured. The terminal was also pre-loaded with the Ca-sensitive dye Furato perform dual wavelength measurements of presynaptic free Ca2+ ion concentration, ([Ca*+]J. Iontophoretic injection of GTPyS into the giant presynaptic terminal caused a marked depression of synaptic transmission. The rate of rise of PSPs began to decrease within a few minutes after the onset of injection and continued progressively for several tens of minutes after injection has been stopped. One hundred microCoulomb injections inhibited transmission by 60-80%. An upper estimate of the intraterminal concentration of injected nucleotide is in the high micromolar range. Additional injections did not seem to add to the inhibition. Inhibition of synaptic transmission was not accompanied by any significant changes in the electrical properties of the presynaptic terminal: neither presynaptic resting nor action potentials were affected by GTPyS injections. Further, the changes in the presynaptic [Ca2’li induced by a train of 250 action potentials (5s 50 Hz) were not affected by injection of GTPyS. There was also no steady depolarization of the postsynaptic membrane potential, suggesting that GTPyS did not increase the tonic release of MPSPs. Therefore, GTPrS acts by altering the amount of transmitter release evoked by a given rise in intracellular calcium, rather than by regulating Ca2+ entry into the terminal. We also evaluated the possibility that a low molecular weight GTP-binding protein is involved in this inhibition. A nonhydrolyzable analogue of GDP, GDPPS, was therefore injected into the terminal. GDPS i.e., visible inhibition of synaptic transmission required injections lasting about five-fold longer than those of GTPyS (about 30 min). This could be due to the lower electric charge of GDPpS (-3 as compared to -4 for GTP$S) causing a smaller quantity of the nucleotide being inotophoretitally injected. Alternatively, GDPpS could be weaker or slower in inhibiting release. Like GTPyS, GDP/IS had no significant effect on presynaptic potentials or [Ca2+]i signals. 1.4 I I