The inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are a family of Ca2+ release channels localized predominately in the endoplasmic reticulum of all cell types. They function to release Ca2+ into the cytoplasm in response to InsP3 produced by diverse stimuli, generating complex local and global Ca2+ signals that regulate numerous cell physiological processes ranging from gene transcription to secretion to learning and memory. The InsP3R is a calcium-selective cation channel whose gating is regulated not only by InsP3, but by other ligands as well, in particular cytoplasmic Ca2+. Over the last decade, detailed quantitative studies of InsP3R channel function and its regulation by ligands and interacting proteins have provided new insights into a remarkable richness of channel regulation and of the structural aspects that underlie signal transduction and permeation. Here, we focus on these developments and review and synthesize the literature regarding the structure and single-channel properties of the InsP3R.

SUMMARY Mitochondrial Ca2+ (Ca2+m) uptake is mediated by an inner membrane Ca2+ channel called the uniporter. Ca2+ uptake is driven by the considerable voltage present across the inner membrane (ΔΨm) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca2+ concentration is maintained 5–6 orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca2+]m under basal conditions. In its absence, mitochondria become constitutively loaded with Ca2+, triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca2+ threshold for Ca2+m uptake without affecting the kinetic properties of MCU-mediated Ca2+ uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca2+m uptake that is essential to prevent [Ca2+]m overload and associated stress.

Familial Alzheimer's disease (FAD) is caused by mutations in amyloid precursor protein or presenilins (PS1, PS2). Many FAD-linked PS mutations affect intracellular calcium (Ca 2+ ) homeostasis by mechanisms proximal to and independent of amyloid production, although the molecular details are controversial. Here, we demonstrate that several FAD-causing PS mutants enhance gating of the inositol trisphosphate receptor (InsP 3 R) Ca 2+ release channel by a gain-offunction effect that mirrors the genetics of FAD and is independent of secretase activity. In contrast, wild type PS or PS mutants that cause frontotemporal dementia have no such effect. FAD PS alter InsP 3 R channel gating by modal switching. Recordings of endogenous InsP 3 R in lymphoblasts derived from individuals with FAD or cortical neurons of asymptomatic PS1-AD mice revealed they have higher occupancy in a high open probability burst mode compared to that of InsP 3 R in cells with wild-type PS, resulting in enhanced Ca 2+ signaling. These results indicate that exaggerated Ca 2+ signaling through InsP 3 R-PS interaction is a disease-specific and robust proximal mechanism in FAD.

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is an endoplasmic reticulum–localized Ca2+-release channel that controls complex cytoplasmic Ca2+ signaling in many cell types. At least three InsP3Rs encoded by different genes have been identified in mammalian cells, with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. To examine regulation of channel gating of the type 3 isoform, recombinant rat type 3 InsP3R (r-InsP3R-3) was expressed in Xenopus oocytes, and single-channel recordings were obtained by patch-clamp electrophysiology of the outer nuclear membrane. Gating of the r-InsP3R-3 exhibited a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). In the presence of 0.5 mM cytoplasmic free ATP, r-InsP3R-3 gating was inhibited by high [Ca2+]i with features similar to those of the endogenous Xenopus type 1 InsP3R (X-InsP3R-1). Ca2+ inhibition of channel gating had an inhibitory Hill coefficient of ∼3 and half-maximal inhibiting [Ca2+]i (K inh) = 39 μM under saturating (10 μM) cytoplasmic InsP3 concentrations ([InsP3]). At [InsP3] < 100 nM, the r-InsP3R-3 became more sensitive to Ca2+ inhibition, with the InsP3 concentration dependence of K inh described by a half-maximal [InsP3] of 55 nM and a Hill coefficient of ∼4. InsP3 activated the type 3 channel by tuning the efficacy of Ca2+ to inhibit it, by a mechanism similar to that observed for the type 1 isoform. In contrast, the r-InsP3R-3 channel was uniquely distinguished from the X-InsP3R-1 channel by its enhanced Ca2+ sensitivity of activation (half-maximal activating [Ca2+]i of 77 nM instead of 190 nM) and lack of cooperativity between Ca2+ activation sites (activating Hill coefficient of 1 instead of 2). These differences endow the InsP3R-3 with high gain InsP3–induced Ca2+ release and low gain Ca2+–induced Ca2+ release properties complementary to those of InsP3R-1. Thus, distinct Ca2+ signals may be conferred by complementary Ca2+ activation properties of different InsP3R isoforms.

The Rh blood group proteins are well known as the erythrocyte targets of the potent antibody response that causes hemolytic disease of the newborn. These proteins have been described in molecular detail; however, little is known about their function. A transport function is suggested by their predicted structure and from phylogenetic analysis. To obtain evidence for a role in solute transport, we expressed Rh proteins in Xenopus oocytes and now demonstrate that the erythroid Rh-associated glycoprotein mediates uptake of ammonium across cell membranes. Rh-associated glycoprotein carrier-mediated uptake, characterized with the radioactive analog of ammonium [ 14 C]methylamine (MA), had an apparent EC 50 of 1.6 mM and a maximum uptake rate (V max ) of 190 pmol/oocyte/min. Uptake was independent of the membrane potential and the Na ؉ gradient. MA transport was stimulated by raising extracellular pH or by lowering intracellular pH, suggesting that uptake was coupled to an outwardly directed H ؉ gradient. MA uptake was insensitive to additions of amiloride, amine-containing compounds tetramethyl-and tetraethylammonium chloride, glutamine, and urea. However, MA uptake was significantly antagonized by ammonium chloride with inhibition kinetics (IC 50 ‫؍‬ 1.14 mM) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H ؉ -coupled counter-transport mechanism.The human Rh blood group proteins have been known for decades to cause hemolytic disease of the newborn, which can result in severe fetal morbidity and mortality (1). Despite their clinical importance, these multipass membrane proteins were not successfully isolated until the late 1980s (2), and little progress was made in their characterization until the genes were cloned (3). Database searches for protein sequences with similarities to Rh proteins were not informative until the Caenorhabditis elegans sequencing initiative revealed that homologs exist in that species. These homologs, in turn, revealed a distant similarity between Rh proteins and ammonium transporters from bacteria and yeast. Since then additional Rh homologs have been found in many organisms (4), and nonerythroid Rh homologs were detected in human and mouse kidney, testis, brain, and liver (5, 6).The erythrocyte Rh blood group antigens are carried on two 417-amino acid polypeptides, RhD and RhCE, which are 97% identical. Rh-negative individuals carry a deletion or mutation in RHD and lack RhD protein (for reviews, see Refs. 7 and 8). A related 409-amino acid Rh-associated glycoprotein (RhAG)