FAST ACTIVATION AND INACTIVATION OF INOSITOL TRISPHOSPHATE-EVOKED CA2+ RELEASE IN RAT CEREBELLAR PURKINJE NEURONS

1. Calcium release from stores via inositol trisphosphate (InsP(3)) activation of intracellular Ca2+ receptor-channels is thought to have a role in regulating the excitability of cerebellar Purkinje neurones. The kinetic characteristics of InsP(3) receptor activation in Purkinje neurones are reported here. 2. InsP(3) was applied by flash photolysis of caged InsP(3) during whole-cell patch clamp. Ca2+ flux into the cytosol was measured with a loci-affinity fluorescent Ca2+ indicator and by activation of Ca2+-dependent membrane conductance. 3. InsP(3) produced Ca2+ release from stores with an initial well-defined delay (mean, 85 ms at 10 mu M InsP(3)), which decreased to less than 20 ms at high InsP(3) concentrations. 4. The rate of rise of free [Ca2+], which provides a measure of Ca2+ efflux and InsP(3) receptor activation, increased with increasing InsP(3) concentration in each cell and had a high absolute value of up to 1400 mu M s(-1) at 40 mu M InsP(3). The period of fast efflux was brief, inactivating in 25 ms at low and in 9 ms at high InsP(3) concentration. 5. Peak free [Ca2+] was high (mean, 23 mu M with a pulse of 40 mu M InsP(3)) and increased with InsP(3) concentration up to 80 mu M InsP(3) tested here. 6. Experiments with a flash-released, stable 5-thio-InsP(3), confirm that the low InsP(3) sensitivity of Purkinje neurones does not result from metabolism of InsP(3). 7. The low functional affinity and fast activation by InsP(3) suggest a difference in InsP(3) receptor properties from non-neuronal cells tested in the same way. The large Ca2+ efflux and high peak [Ca2+] probably result from high InsP(3) receptor-channel density. 8. Elevated cytosolic [Ca2+] produced by Ca2+ influx through plasmalemmal Ca2+ channels strongly suppressed InsP(3)-evoked Ca2+ release from stores. Rapid termination of InsP(3)-evoked efflux results mainly from inhibition by high [Ca2+]. 9. The fast InsP(3) activation kinetics and rapid, strong inactivation by Ca2+ influx suggest that interactions between InsP(3)-mediated and membrane Ca2+ signalling could occur on a time scale compatible with neuronal excitation.