Inositol 1,4,5-trisphosphate receptors in the endoplasmic reticulum: A single-channel point of view

Abstract

As an intracellular Ca(2+) release channel at the endoplasmic reticulum membrane, the ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) plays a crucial role in the generation, propagation and regulation of intracellular Ca(2+) signals that regulate numerous physiological and pathophysiological processes. This review provides a concise account of the fundamental single-channel properties of the InsP3R channel: its conductance properties and its regulation by InsP3 and Ca(2+), its physiological ligands, studied using nuclear patch clamp electrophysiology.

Keywords: Calcium signal; Endoplasmic reticulum; Inositol 1,4,5-trisphosphate receptor; Intracellular calcium; Ion channel; Patch clamp electrophysiology.

Fig. 1. Different configurations for nuclear patch clamping experiments

Schematic diagrams illustrating the orientation of InsP₃R channels in nuclear membrane patches in various configurations of nuclear patch-clamp experiments. (A) On-nucleus configuration with outer nuclear membrane intact, (B) excised luminal-side-out configuration, (C) excised cytoplasmic-side-out configuration, (D) on-nucleus configuration with outer nuclear membrane removed, (E) excised nucleoplasmic-side-out configuration. (F) Diagram showing how the two aspects of the InsP₃R channel are represented in this figure. Figure modified from [19].

Fig. 2. Measurement of unitary Ca²⁺ current through a type 3 InsP₃R channel in physiological ionic conditions

(A) Linear fits (purple and red lines) to selected open-channel current (I_open) and closed-channel leak current (I_closed) vs. V_app data (blue and pink points, respectively) from a single-channel nuclear patch clamp experiment in excised lum-out configuration under symmetric ionic conditions with pipette and perfusion solutions containing 140 mM KCl, 0.5 mM MgCl₂, and 3 μM free [Ca²⁺]. Pipette solution contained 2 μM InsP₃. (B) Fitted I_open-V_app and I_closed-V_app lines in I-V_app region marked by the black rectangle in (a). Zero-current level (black dotted line) established at the intersection of the I-V_app fits at V_app = 0 (marked by orange arrowhead). (C) Linear fits to I_open and I_closed data from V_app ramps recorded for the same membrane patch under asymmetric ionic conditions with perfusion solution containing 2 mM CaCl₂. Color and symbol conventions, I and V_app ranges are the same as in (a). (D) Fitted I–V lines in the same I–V region and with the same zero-current level as (b). V_app = 0 (marked by orange arrowhead) at the intersection of the I_closed-V_app line and zero-current level. Unitary Ca²⁺ current is I_open at V_app = 0 (marked by green double arrow). Figure modified from [52].

Fig. 3. Ligand dependencies of gating of homotetrameric recombinant InsP₃R channels of various isoforms expressed in DT40-3KO cells

(A) Typical single-channel on-nucleus patch-clamp current traces of InsP₃R-3 channels in sub-optimal (190 nM), optimal (2μM) and inhibitory (23 μM) [Ca²⁺]_i in the presence of saturating (10 μM) [InsP₃]. V_app = –40 mV. Arrow indicates closed channel baseline current level for these and all subsequent current traces. (B–D) [Ca²⁺]_i-dependencies of mean channel P_o in various [InsP₃] and [ATP⁴⁻] as tabulated, for rat InsP₃R-1, mouse InsP₃R-2 and rat InsP₃R-3 channels, respectively. Curves are fitted to mean P_o data points using the empirical biphasic Hill equation with 5 parameters [11]. Data in (b) and (c) are from [50]. (a) and (d) are modified from [57].

Fig. 4. Effects of [Ca²⁺]_ER on the gating of homotetrameric InsP₃R-3 channels expressed in DT40-3KO cells

Typical current traces from nuclear patch clamp experiments in lum-out configuration under experimental conditions ([InsP₃], [Ca²⁺]_i, concentrations of Ca²⁺ chelator in pipette solutions solution and V_app) tabulated at top. Color bars at top indicate changes in [Ca²⁺]_ER achieved by rapid perfusion techniques. Blue bars at bottom mark the part of the current traces used to evaluate the channel P_o tabulated below. Figures are modified from [57]. For (G), V_app should be +70 mM.

Fig. 5. Modal gating behaviors of endogenous insect Sf9 InsP₃R

(A) Black traces are baseline-subtracted current records obtained in saturating 10 μM InsP₃, with sub-optimal (100 nM), optimal (1 μM) and inhibitory (89 μM) [Ca²⁺]_i, as tabulated. Blue and purple traces are idealized gating records before and after burst analysis, respectively, derived from the corresponding current records. Color bars at bottom indicate the gating mode the InsP₃R channel was in during the shown records (red, orange and green for L, I and H modes, respectively). (B) Channel open probability in each of the three modes (P_o^M), (C) probability of the channel being in each of the three modes (π^M), and (D) mean durations 〈τ^M〉 of each of the three modes observed are plotted against the ligand concentrations in which they were observed. Note the non-linear y-axis used in (B) to better show the low P_o^L values for the L mode, and the logarithmic scale used in (D). In (B-D), the top x-axis labels indicate the cytoplasmic free calcium concentrations, the bottom labels indicate the cytoplasmic InsP₃ concentrations. Figure modified from [12] and [73].

Fig. 6. Dynamic responses of endogenous insect Sf9 InsP₃R to abrupt changes in [Ca²⁺]_i and [InsP₃] observed in cyto-out nuclear patch clamp experiments

Color bars at top indicate changes in [Ca²⁺]_i and [InsP₃] achieved by rapid exchange of bath solutions. Times when such exchanges occurred were marked by changes in closed-channel current levels due to difference in [KCl] (100 mM vs. 70 mM) in the bath solutions used. Gray bars at bottom indicate the lag times between ligand concentration switches and the resulting changes in InsP₃R channel gating behaviors. Two arrows indicate the closed-channel current levels before and after the bath solution switches for each current traces. In (J), the dark gray bar indicates the Ca²⁺ activation lag time and the light gray bar indicates the Ca²⁺ inhibition lag time. Note that a different time scale is used for (L) and (M). Figure modified from [77].