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Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors.
Mak DO, McBride S, Foskett JK.
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The inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP3R) is an endoplasmic reticulum-localized Ca2+ -release channel that controls complex cytoplasmic Ca(2+) signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 Ins3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of approximately 3 and half-maximal inhibiting [Ca2+]i (Kinh) = 39 microM under saturating (10 microM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP(3) concentration dependence of Kinh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of approximately 4. InsP(3) activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3-induced Ca2+ release and low gain Ca2+ -induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.
Figure 1. Expression of endogenous X-InsP3R-1 and recombinant r-InsP3R-3 in cRNA-injected (+) and control, uninjected (−) Xenopus oocytes. Western analysis was performed as described in Mak et al. 2000. (A–H) Immunoblotted with InsP3R-1-specific antibody (Joseph and Samanta 1993; Joseph et al. 1995); (I–J) immunoblotted with InsP3R-3-specific antibody (Transduction Labs.). Aliquots equivalent to n oocytes from the same lysate sample were used in the lanes.
Figure 2. Typical single-channel current traces of the r-InsP3R-3 at various [Ca2+]i in the presence of 10 μM InsP3. Arrows indicate closed channel current level in all current traces.
Figure 3. Ca2+ dependencies of mean open channel duration (Α) and closed channel duration (B) of the r-InsP3R-3 activated by various concentrations of InsP3 as tabulated. In the closed channel duration graph, data points obtained with the same InsP3 concentration are connected with a line for clarity.
Figure 4. Ca2+ dependence of r-InsP3R-3 channel open probability under various [InsP3]. Different symbols denote data for various [InsP3] as tabulated. The curves are theoretical fits using the Hill equation (), with Kinh varying with [InsP3] as listed in the graph, whereas Pmax, Kact, Hact, and Hinh remained independent of [InsP3] with values tabulated in the graph.
Figure 5. Single-channel open and closed channel duration histograms of r-InsP3R-3 in 10 μM InsP3, with 29 nM, 224 nM, 1.2 μM, and 57.5 μM Ca2+, respectively, as listed in the graphs. The open and closed duration histograms shown for each [Ca2+]i were derived from one patch-clamp experiment performed under the stated [Ca2+]i. Similar histograms were obtained from two additional experiments at each set of experimental conditions. The smooth curves are theoretical probability density functions consisting of two to four exponential components fitted to the histograms, as outlined in Sigworth and Sine 1987. The time constant and relative weight of each exponential component is labeled besides the corresponding peak in the curves. Duration histograms obtained with the same set of experimental conditions are fitted with the same number of exponential components and the time constants and relative weights of corresponding exponential components lie within ∼30% of the values shown.
Figure 6. Comparison of the Ca2+ dependencies of Po of r-InsP3R-3 and X-InsP3R-1 in 10 μM InsP3 and 0.5 mM ATP. Open circles represent data for r-InsP3R-3 from this study, fitted with the solid curve; closed circles represent data for X-InsP3R-1 taken from (Mak et al. 1998), fitted with the dashed curve. The curves are calculated using the Hill equation () with the tabulated parameters. Higher affinity and lack of cooperativity of the Ca2+ activation sites of the type 3 channel endow it with high gain IICR and low gain CICR. In contrast, lower affinity and presence of cooperativity of the Ca2+ activation sites of the type 1 channel confer low gain IICR and high gain CICR. Under resting [Ca2+]i, low levels of stimulation will trigger release of Ca2+ by IICR from the type 3 channel, which in turn will trigger further release by CICR from the type 1 channel.
Figure 7. Single-channel open and closed channel duration histograms and the fitted theoretical probability density functions of X-InsP3R-1 in 10 μM InsP3 with 31 and 224 nM Ca2+, respectively, obtained as described in the legend to Fig. 5.
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