Fast Biphasic Regulation of Type 3 Inositol Trisphosphate Receptors by Cytosolic Calcium

In cytosol-like medium (CLM) with a free [Ca2+] of 200 nm, a supramaximal concentration of inositol 1,4,5-trisphosphate (IP3) (30 μm) evoked 45Ca2+ release from type 3 IP3 receptors only after a latency of 48 ± 6 ms; this latency could not be reduced by increasing the IP3concentration. In CLM containing a low free [Ca2+] (∼4 nm), 300 μm IP3 evoked45Ca2+ release after a latency of 66 ± 11 ms; this was reduced to 14 ± 3 ms when the [Ca2+] was 1 mm. Preincubation with CLM containing 100 μm Ca2+ caused a rapid (half-time = 33 ± 9 ms), complete, and fully reversible inhibition that could not be overcome by a high concentration of IP3 (300 μm). Hepatic (type 2) IP3 receptors were not inhibited by Ca2+ once they had bound IP3, but 100 μm Ca2+ rapidly inhibited type 3 IP3 receptors whether it was delivered before addition of IP3 or at any stage during a response to IP3. Ca2+ increases the affinity of IP3 for hepatic receptors by slowing IP3 dissociation, but Ca2+had no effect on IP3 binding to type 3 receptors. The rate of inhibition of type 3 IP3 receptors by Ca2+was faster than the rate of IP3 dissociation, and occurred at similar rates whether receptors had bound a high (adenophostin) or low affinity (3-deoxy-3-fluoro-IP3) agonist. Dissociation of agonist is not therefore required for Ca2+ to inhibit type 3 IP3 receptors. We conclude that type 2 and 3 IP3 receptors are each biphasically regulated by Ca2+, but by different mechanisms. For both, IP3 binding causes a stimulatory Ca2+-binding site to be exposed allowing Ca2+ to bind and open the channel. IP3 binding protects type 2 receptors from Ca2+ inhibition, but type 3 receptors are inhibited by Ca2+ whether or not they have IP3 bound. Increases in cytosolic [Ca2+] will immediately inhibit type 3 receptors, but inhibit type 2 receptors only after IP3 has dissociated.