IP3 Receptor Channels are Clustered Before IP3 Exposure with No Discernible Effect on Single-Channel Gating Properties

Abstract

A recent patch clamp study reported that recombinant rat type 3 IP3R Ca2+ release channels expressed in IP3R deficient DT40-KO cells are randomly distributed in the ER/outer nuclear membrane until exposure to low [IP3] induces their rapid clustering [Rahman et al (2009) Nature458: 655-659]. In sub-optimal ligand conditions, clustered channels gated identically and independently, but with a lower open probability (Po) than lone channels regardless of cluster size. In contrast, in optimal ligand conditions, Po for clustered and lone channels were the same, but positive cooperative gating was consistently detected in patches with two active channels. Seeking to verify these surprising observations, we acquired current records from the same channels expressed in the same cell system with identical protocols and ligand conditions. The records were analyzed with the same algorithm to characterize gating behaviors of lone and clustered channels separately. For comparison, all nuclear patch-clamp current records previously acquired under comparable ligand conditions for recombinant rat type 3 IP3R channels expressed in Xenopus oocytes, endogenous Xenopus type 1 IP3R channels in oocytes and endogenous IP3R channels in insect Sf9 cells were similarly analyzed. We found that rat type 3 IP3R channels in DT40-KO cells are clustered without exposure to IP3, like all IP3R channels investigated before. For all IP3R channels examined, we detected no significant differences between channel Po of lone and clustered channels, in sub-optimal or optimal ligand conditions. Furthermore, in two-channel current records, the same pattern of channel gating was detected, in both sub-optimal and optimal ligand conditions, with only a small fraction (< 15%) revealing positive cooperative gating behavior. Thus, single channel behavior in all ligand conditions is independent of whether the IP3R channel is in single or multi-channel patches.