The ubiquitous endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) channel raises cytoplasmic free Ca2+ concentration ([Ca2+]i) in response to extracellular stimuli by releasing Ca2+ stored in the ER lumen to generate and modulate complex Ca2+ signals that control numerous physiological processes, from apoptosis and secretion to immune responses and memory. Although regulation of InsP3R channel activity by cytoplasmic [InsP3] and [Ca2+]i has been well studied, the effects of physiological changes in free [Ca2+] in the ER lumen ([Ca2+]ER) on InsP3R channel activity remain poorly understood and controversial. Better understanding of such effects will provide insights into the connections between [Ca2+]ER homeostasis and proper functioning of InsP3R-modulated intracellular physiological processes including regulation of cell bioenergetics and protein biogenesis; and between [Ca2+]ER dysregulation and pathogenesis of conditions like ER stress responses, diabetes, cardiac dysfunction, defective cell proliferation and cell death. Previous studies of [Ca2+]ER effects on InsP3R channel activity used mostly 45Ca2+ flux measurement and fluorescence Ca2+ imaging, both of which infer InsP3R channel activity from changes in [Ca2+]i or [Ca2+]ER, and therefore cannot rigorously control both [Ca2+]ER and [Ca2+]i simultaneously. To provide insights into [Ca2+]ER modulation of InsP3R channel activity, we have investigated, in preliminary studies, single InsP3R channel activities under rigorously controlled ionic conditions by the nuclear patch clamp method that we pioneered. Activities of single InsP3R channels were continuously monitored as [Ca2+]ER was changed by rapid perfusion solution switches or by modulating activity of SERCA at the outer nuclear membrane. We have discovered that [Ca2+]ER modulates InsP3R channel gating by two distinct mechanisms: (1) driving Ca2+ flux through the open channel to raise local [Ca2+]i in the vicinity of the channel, thereby altering its activity through its cytoplasmic Ca2+-sensing sites; and (2) a profound Ca2+-flux-independent effect mediated likely by a peripheral accessory protein(s) localized to the ER lumen in many cell types. We will apply single-channel nuclear electrophysiology and [Ca2+]i imaging to systematically quantify each of these effects individually in various [Ca2+]i, [InsP3], and [Ca2+]ER for various kinds of InsP3R channels (recombinant and endogenous, homo- and hetero-tetrameric ones of different InsP3R isoforms). Furthermore, our preliminary studies demonstrated that members of the annexin and copine families of proteins are present in the ER lumen and interact specifically, in a [Ca2+]ER dependent manner, with a ER luminal fragment conserved in all InsP3R isoforms. We will verify, using molecular biological and electrophysiological methods the roles of these proteins in mediating the Ca2+-flux independent [Ca2+]ER effect on single InsP3R channel activities, and in regulating Ca2+ signaling at the cellular level. These studies will provide novel insights into [Ca2+]ER regulation of InsP3R channel activity, which appears to be as profound as the better studied [Ca2+]i regulation of the channel.