InsP₃R channel gating altered by clustering?

The inositol trisphosphate receptor (InsP₃R) forms a calcium channel that resides in the membrane of the endoplasmic reticulum and is activated by inositol trisphosphate (InsP₃). InsP₃ is a phosphorylated monosaccharide that is generated via hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and activation of the InsP₃R is involved in a broad range of biological processes, including cell division, apoptosis and development. Rahman et al.^1,2 reported that exposure to low concentrations of InsP₃ induces rapid clustering of InsP₃R Ca²⁺ release channels normally randomly distributed in endoplasmic reticulum/outer nuclear membranes. Importantly, clustered channels gate differently from lone channels. Using similar protocols, we observed InsP₃R channel clustering without exposure to InsP₃ (Fig. 1a), as we found in other systems^3–5 with protocols designed to avoid InsP₃ pre-exposure. More significantly, we find that clustering has no effect on InsP₃R channel gating. For this reason, we believe that InsP₃-induced channel clustering and modification of channel gating by clustering may not be universal phenomena.

Figure 1. InsP₃R channels are clustered before exposure to InsP₃, with gating properties unaltered by clustering.

a, N_A in nuclear membrane patches with no pre-exposure to InsP₃ obtained from InsP₃R3 expressing DT40-KO cells. Note nonlinear square-root scale for frequency axis. b, P_o observed under saturating [InsP₃] and sub-optimal [Ca²⁺]_i in multi- and apparent single-channel current records for recombinant rat InsP₃R3 (r-3) channels expressed in DT40-KO (DK) cells or Xenopus oocytes (Xo), endogenous Xenopus InsP₃R1 (X-1) channels from Xenopus oocytes (Xo), and endogenous insect InsP₃R (iR) channels from Sf9 cells. Concentrations of free ATP⁴⁻ ([ATP]_f) in the pipette solutions used are indicated. Mean P_o with s.e.m. (as error bars) and P_o for individual current records are shown, together with mean P_o from Rahman et al.¹ c, P_o of r-3 channels in DK cells in optimal [Ca²⁺]_i and sub-saturating [InsP₃], ligand conditions not investigated in Rahman et al.¹. Same symbols as in b are used.

Rahman et al.^1,2 reported that in sub-optimal cytoplasmic free Ca²⁺ concentrations ([Ca²⁺]_i), clustered recombinant rat type 3 InsP₃R (InsP₃R3) channels expressed in InsP₃R-deficient DT40-KO cells gated identically and independently, but with lower open probability (P_o) than lone channels, regardless of cluster size. In contrast, clustered channels had the same P_o as lone channels in optimal ligand conditions, but gated with positive cooperativity. If broadly observed, these surprising findings have important implications for understanding InsP₃-mediated Ca²⁺ signals, and for quantitative analyses in single-channel InsP₃R electrophysiology.

To verify these observations, we examined the same InsP₃R3 channels in the same DT40-KO cells using similar protocols and ligand conditions. Specifically, we used 5 mM (same as Rahman et al.¹) and 0.5 mM (more physiological) cytoplasmic free [ATP⁴⁻] ([ATP]_f). Records with ≤4 active channels were analysed with the same algorithm¹. In addition, we similarly analysed nuclear patch-clamp records previously acquired under comparable ligand conditions for recombinant rat InsP₃R3 expressed in Xenopus oocytes⁶, endogenous Xenopus type 1 InsP₃R (InsP₃R1) in oocytes⁷ and endogenous insect InsP₃R in Sf9 cells⁵. For all channels examined in these various systems, we detected no statistical difference (P > 0.05, t-test) between P_o in single- versus multi-channel patches in saturating [InsP₃] and sub-optimal [Ca²⁺]_i (Fig. 1b), or in sub-saturating [InsP₃] and optimal [Ca²⁺]_i (Fig. 1c). Furthermore, in two-channel records, similar channel gating patterns were detected in all [Ca²⁺]_i (Fig. 2), with only a small fraction exhibiting positive cooperativity. Thus, our extensive data set reveals no effect of clustering on InsP₃R channel gating in all ligand conditions.

Figure 2. Distribution of cooperativity index for two-channel current records of different InsP₃R channels in various systems in optimal and sub-optimal [Ca²⁺]_i.

Filled and open circles represent records with two channels exhibiting identical and independent, or non-binomial gating, respectively. Non-binomial records with cooperativity index, (P₂+P₁/2)²−P₂, significantly greater than 0 (in yellow shaded region) had two channels gating with different P_o, and those with cooperativity index significantly smaller than 0 (in blue shaded region) had two channels gating with positive cooperativity. The cooperativity indices have no correlation with the durations of the current records (data not shown) and therefore are unlikely to be significantly affected by current record durations limited by channel inactivation.

In constant ligand conditions, we consistently observed abrupt, stochastic, irreversible inactivation of InsP₃R in on-nucleus or excised luminal-side-out nuclear patches, with mean activity durations of ~40 s for oocyte InsP₃R (ref. 3), ~100 s for Sf9 InsP₃R (ref. 5) and ~140 s for InsP₃R from DT40-KO cells, whereas Rahman et al. reported no such inactivation¹. Importantly, we analysed only current records long enough for the number of active channels to be counted with >99% confidence^1,5,6,8. Because finite time elapsed between pipettes making contact with the outer nuclear membrane and gigaohm seal formation (< 5 s for oocyte and Sf9 nuclei, ~10 s for DT40 nuclei), apparent single-channel patches possibly included a fraction (~11–26%) that actually contained multiple channels in which all but one channel inactivated before gigaohm seal formation. Yet, the mean number of active channels (N_A) we observed for InsP₃R3 in DT40 nuclear patches (1.36 ± 0.12 for 211 patches) is similar to that reported in Rahman et al.¹, suggesting that inactivation did not substantially impair our ability to count channels in these patches. We did detect larger N_A (10.8 ± 1)⁸ in outside-out nuclear patches, which probably have significantly larger membrane areas and were isolated using a different technique, and therefore are not an appropriate comparison to illustrate either the variability in InsP₃R expression level in DT40-KO cells or the effect of inactivation on N_A detected.

The P_o distributions that we observed in apparent single-channel and true multi-channel patches were similar, with no indication that two populations of channels with different P_o exist. Furthermore, mean P_o of our true multi-channel patches is comparable to the lone-channel P_o observed by Rahman et al.¹ (Fig. 1b). Thus, our conclusion that clustering does not affect InsP₃R channel gating is not compromised by the irreversible inactivation of InsP₃R channels.

We have no clear explanation for the discrepancies between our observations and those reported by Rahman et al.¹. However, neither InsP₃-induced InsP₃R clustering nor its modification of InsP₃R gating are consistently observed for InsP₃R expressed in DT40-KO and other cells. In contrast, channel clustering before InsP₃ exposure was observed in all cell systems investigated without effect on channel gating^3–5. Thus, we suggest that InsP₃-induced channel clustering and modification of channel gating by clustering may not be universal phenomena.

METHODS SUMMARY

Single-channel P_o for a sufficiently long⁵ current record with N_A active channels was evaluated as $={\displaystyle {\sum }_{i=0}^{N_{\mathrm{A}}}[i{P}_i]}/N_{\mathrm{A}}$, where P_i, the probability that i channels were active simultaneously in the record, was determined by the same method as Rahman et al.¹. The channel gating pattern for a two-channel current record was determined from P_i. If P_i are similar to the expected binomial values, $\left[2!P_{\mathrm{o}}{}^i{(1-P_{\mathrm{o}})}^{2-i}\right]/[i!(2-i)!]$ for i = 0,1,2 (P > 0.05 by χ²-test), the channels gated independently with similar P_o. Otherwise, if the cooperativity index, (P₂+P₁/2)² − P₂, is >0, they gated with different P_o, with or without negative cooperativity. If (P₂+P₁/2)² − P₂ < 0, they gated with positive cooperativity⁹.