Calcium-Flux-Mediated Modulation of InsP3R Channel Activity by ER Luminal Calcium Concentration

Abstract

The ubiquitous, mostly endoplasmic reticulum (ER)-localized InsP3R channel modulates cytoplasmic free [Ca2+] ([Ca2+]i) to generate complex Ca2+ signals by releasing Ca2+ stored in the ER lumen. Whereas cytoplasmic InsP3 activation and biphasic [Ca2+]i regulation of the channel have been well studied, its regulation by ER luminal free [Ca2+] ([Ca2+]ER) is poorly understood and controversial. Here, we used excised luminal-side-out nuclear patch clamping with perfusion solution exchange to study the effects of [Ca2+]ER on homotetrameric rat type 3 InsP3R (InsP3R-3) channel activity. In optimal 2 μM [Ca2+]i (buffered by 0.5 mM diBrBAPTA) and sub-saturating 3 μM InsP3, jumps of [Ca2+]ER from 70 nM (no Ca2+ flux through channel) to 300 μM (substantial Ca2+ flux) reduced channel open probability (Po) significantly. This inhibition was abrogated when saturating 10 μM InsP3 was used, but was restored when [Ca2+]ER was raised to 2 mM. This [Ca2+]ER effect exhibited a biphasic dependence on [Ca2+]i because jumps of [Ca2+]ER (70 nM to 300 μM) in 3 μM InsP3 increased Po in sub-optimal 70 nM Ca2+i. These effects of [Ca2+]ER were attenuated when an electrical potential was applied to oppose Ca2+ flux through the channel. Importantly, the effects of [Ca2+]ER on channel activity depended on cytoplasmic Ca2+ buffering conditions: it was stronger when [Ca2+]i was weakly buffered by 0.1 mM HEDTA (a slow Ca2+ chelator) but was completely abolished with 5 mM diBrBAPTA buffering. These observations are completely accounted for by Ca2+ flux driven by [Ca2+]ER through the open InsP3R channel raising local [Ca2+]i around the channel to regulate its Po through its cytoplasmic activating and inhibitory Ca2+-binding sites. These results provide no evidence for a role of [Ca2+]ER in regulating InsP3R-3 activity by effects mediated directly on the luminal aspect of the channel.