Inositol (1,4,5)-trisphosphate (InsP₃)-gated Ca channels from Cerebellum: Conduction properties for divalent cations and regulation by intraluminal calcium

The conduction properties of inositol (1,4,5)-trisphosphate (InsP₃)-gated calcium (Ca) channels (InsP₃R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single-channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP₃R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (∼5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single-ion pores. Measurements of InsP₃R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (≥ 10 mM) decreased channel open probability 5-10-fold. When InsP₃R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a P_Ba/P_K of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP₃R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP₃R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltagegated Ca channels of the plasma membrane, (b) The estimated size of the Ca current through the InsP₃R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP₃R by intraluminal Ca in the submillimolar range remains controversial, (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP₃R activity was developed and the kinetic parameters of InsP₃R gating were determined.