Single-channel properties in endoplasmic reticulum membrane of recombinant type 3 inositol trisphosphate receptor

Abstract

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP(3)R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP(3)R-3 are strikingly similar to those of Xenopus type 1 InsP(3)R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP(3)R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP(3)R isoforms is a mechanism to modify the temporal and spatial features of [Ca(2+)](i) signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP(3)Rs that have relatively similar Ca(2+) permeation properties.

Figure 1

Expression of endogenous type 1 and recombinant type 3 InsP₃ receptors in Xenopus oocytes. (A–F) Immunoblotted with Ab-3; (G–K) immunoblotted with Ab-1. (A) Western blot of lysate from an equivalent of one control oocyte, demonstrating lack of endogenous type 3 InsP₃R expression; (B) Western blot of lysate from an equivalent of one r-InsP₃R-3 cRNA-injected oocyte, demonstrating expression of the recombinant r-InsP₃R-3 protein; (C) Western blot of type 3 InsP₃R adsorbed nonspecifically to agarose beads, from lysate from fifteen control oocytes; (D) Western blot of type 3 InsP₃R in InsP₃R-1 immunoprecipitate, from lysate from fifteen control oocytes; (E) Western blot of type 3 InsP₃R adsorbed nonspecifically to agarose beads, from lysate from fifteen cRNA-injected oocytes; (F) Western blot of type 3 InsP₃R in InsP₃R-1 immunoprecipitate from lysate, from fifteen cRNA-injected oocytes. Difference between F and E represents the amount of recombinant r-InsP₃R-3 specifically immunoprecipitated with X-InsP₃R-1. (G) Western blot of X-InsP₃R-1 adsorbed nonspecifically to agarose beads, from lysate from fifteen control oocytes; (H) Western blot of X-InsP₃R-1 in InsP₃R-3 immunoprecipitate, from lysate from fifteen control oocytes. The InsP₃R detected is most likely caused by cross-interaction of the immunoprecipitating Ab-3 with the large amount of endogenous X-InsP₃R-1 present in the lysate from fifteen oocytes. (I) Western blot of X-InsP₃R-1 adsorbed nonspecifically to agarose beads, from lysate from fifteen cRNA-injected oocytes; (J) Western blot X-InsP₃R-1 in InsP₃R-3 immunoprecipitate, from lysate from fifteen cRNA-injected oocytes. Difference in protein amounts between H/I and J represents the amount of X-InsP₃R-1 specifically immunoprecipitated with recombinant r-InsP₃R-3. (K) Western blot of X-InsP₃R-1 in lysate from an equivalent of one control oocyte; (L) Western blot of X-InsP₃R-1 in lysate from an equivalent of one cRNA-injected oocyte. Comparisons of K and L indicate that r-InsP₃R-3 expression does not induce changes in the expression levels of the endogenous X-InsP₃R-1. Western blots of type 1 InsP₃R in InsP₃R-1 immunoprecipitates or type 3 InsP₃R in InsP₃R-3 immunoprecipitates are not shown because too much protein was present in those lanes for the protein bands to be properly detected with the same exposure as the other lanes.

Figure 2

Current traces obtained by patch clamping the outer membrane of isolated oocyte nuclei in symmetric 0-mM Mg²⁺ solutions. All current records shown were obtained with V_app = 20 mV, and with 0.5 mM free ATP and 10 μM InsP₃ in the pipette solution. The arrows indicate the closed channel current level. (A, C, and E) Nuclei from r-InsP₃R-3 cRNA-injected oocytes. (B, D, and F) Nuclei from uninjected oocytes. (A and B) [Ca²⁺]_i = 1,150 nM. (C and D) [Ca²⁺]_i = 80 nM. (E and F) [Ca²⁺]_i = 57.5 μM. The average P _o (±SEM) evaluated for n experiments under the shown experimental conditions are: (A) 0.75 ± 0.03 (n = 16); (B) 0.80 ± 0.02 (n = 8); (C) 0.45 ± 0.07 (n = 4); (D) 0.14 ± 0.03 (n = 7); (E) 0.23 ± 0.07 (n = 4); (F) 0.27 ± 0.11 (n = 6).

Figure 3

Conductance of the recombinant r-InsP₃R-3 in the nuclear membrane. (A) Typical patch clamp current traces for r-InsP₃R-3 channels in symmetric solutions containing 2.5 mM Mg²⁺ and [Ca²⁺]_i = 940 nM. (B) I-V relation for the r-InsP₃R-3 (•) in symmetric 2.5 mM Mg²⁺ solutions. Channel currents were averaged from all channel opening and closing events from five current records (over 100 events in each record). SEM bars are smaller than the symbols. I-V relation for the X-InsP₃R-1 channels in the same ionic conditions (○) obtained previously (Mak and Foskett 1998) is also plotted for comparison. Solid curve is a fifth-order odd polynomial [I = a ₁V_app + a ₃(V_app)³ + a ₅(V_app)⁵] fitted to the r-InsP₃R-3 data under positive V_app. (Dotted line) The slope conductances (dI/dV) of 119 pS at 0 mV and 360 pS at 50 mV. Dashed curve: 0.7× the value of the solid curve.

Figure 5

Channel properties of the r-InsP₃R-3 in symmetric 0 mM Mg²⁺ solutions ([Ca²⁺]_i = 80 nM) under a voltage ramp. (A) The applied voltage ramp used to obtain the current traces. (B) I-V relation obtained from a typical current trace using the voltage ramp. Dotted and dashed lines are the fitted closed and open channel I-V relations, respectively. The channel conductance was evaluated as the difference between the slopes of the two fitted lines. (C) Voltage dependence of P _o of the r-InsP₃R-3 channel. The P _o of the channel observed at various V_app during the voltage ramp was binned and averaged over 28 traces to generate the histogram. SEM of the P _o is also plotted. The horizontal bar indicates the voltage range −15 to 15 mV in which channel openings and closings were difficult to discern, so P _o was underestimated.

Figure 4

r-InsP₃R-3 channel conductance fluctuates in the absence of Mg²⁺. (A) Current trace from a patch with two InsP₃R-3 channels, one of which had fluctuating conductance, in symmetric solutions containing 0 mM Mg²⁺; [Ca²⁺]_i = 221 nM. (B) Current trace showing two InsP₃R-3 channels with conductance fluctuating in concert, with 2.5 mM Mg²⁺ on the pipette side and 0 mM Mg²⁺ on the bath side of the channel; [Ca²⁺]_i = 1,260 nM.

Figure 6

Conductance substates of the InsP₃R channels observed in nuclear patches obtained from r-InsP₃R-3 cRNA-injected oocytes. (A) Current trace showing a transition of an InsP₃R channel from the small (S) conductance state to the main (M) conductance state in symmetric solutions containing 0 mM Mg²⁺; [Ca²⁺]_i = 1,150 nM. (B) Current trace showing an InsP₃R channel in the rare half (H) conductance state in symmetric solutions containing 0 mM Mg²⁺; [Ca²⁺]_i = 255 nM. (C) Current trace showing an InsP₃R channel in the normal and flicker kinetic modes in symmetric 2.5 mM Mg²⁺; [Ca²⁺]_i = 940 nM. F₁ and F₂ denote the conductance states in the flicker kinetic mode.

Figure 9

Store-operated calcium influx across the oocyte plasma membrane. Whole-cell transmembrane conductance of r-InsP₃R-3 cRNA-injected Xenopus oocytes was recorded in two types of TEV experiments. (A) Ca²⁺-activated plasma membrane Cl⁻ channel current after microinjection of InsP₃ in various bathing solutions. Arrows indicate injections of InsP₃ into the oocyte. Horizontal bars indicate exchange of bathing solution by perfusion with a high Ca²⁺ solution HCa96 (H) or a low Ca²⁺ solution LCa96 (L). The magnitudes of the changes in membrane conductance due to plasma membrane Ca²⁺-activated Cl⁻ current response to InsP₃ injection ΔG _IP3 ^Cland due to Ca²⁺ influx through store-operated Ca²⁺ channels ΔG _SOC ^Clare marked. (B) Store-operated Ca²⁺ currents after activation of InsP₃R in various bathing solutions. Horizontal bars indicate exchange of bathing solution with high Ca²⁺ solution HCa96 (H), low Ca²⁺ solution LCa96 (L), and low Ca²⁺ LCa96 containing 1:100 fetal bovine serum (fbs). Arrow indicates microinjection of BAPTA. Magnitude of changes in membrane conductance due to Ca²⁺ influx through store-operated Ca²⁺ channels (ΔG) is indicated.

Figure 7

Ion selectivity of the r-InsP₃R-3. (A) The I-V curve of the InsP₃R-3 in the presence of solutions containing asymmetric K⁺ concentrations. The pipette contained the low K⁺ solution. The bath solution was the standard 140 mM KCl solution with 0 mM Mg²⁺. V_app values were corrected for the liquid junction potential (Neher, 1992) between the asymmetric solutions across the membrane patch. Channel currents were obtained as in Fig. 3. • are data points for r-InsP₃R-3 fitted with the dashed line, giving V_rev = 25.2 ± 0.7 mV. ○ are data points for X-InsP₃R-1 fitted with the solid line, giving V_rev = 25.3 ± 0.2 mV. (B) The I-V curve of InsP₃R-3 in the presence of a [Ca²⁺] gradient. The pipette contained the standard 140 mM KCl solution with 0 mM Mg²⁺ and [Ca²⁺]_i = 24.7 nM. To avoid exposing the oocyte nucleus to high [Ca²⁺], which interferes with formation of giga-ohm seals (Mak and Foskett 1994), an excised patch with InsP₃R activities was first obtained from a nucleus in the standard 140 mM KCl solution (0 mM Mg²⁺). The bath solution was then replaced with the high Ca²⁺ solution by perfusion. V_app values were corrected for liquid junction potential (Neher, 1992) between the asymmetric solutions across the membrane patch. Channel currents were obtained as in Fig. 3. The solid curve is a fifth order polynomial fitted to the data points, giving V_rev = 20.6 ± 0.5 mV.

Figure 8

Histogram analysis of number of current levels (abscissa) observed in 767 nuclear patch clamp experiments using isolated nuclei from r-InsP₃R-3 cRNA-injected oocytes (unshaded histogram with thick outline). Also plotted is the Poisson distribution (shaded histogram with thin outline) with the same mean current level number ≪n≫=2.016per patch. Note the different y-axis scales used in the histogram. The number of patches containing a large number of channels (6–15) exceeds that predicted from Poisson statistics, suggesting that the recombinant type 3 InsP₃R channel localizes in clusters.