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.

Stromal interaction molecule (STIM) proteins function in cells as dynamic coordinators of cellular calcium (Ca2+) signals. Spanning the endoplasmic reticulum (ER) membrane, they sense tiny changes in the levels of Ca2+ stored within the ER lumen. As ER Ca2+ is released to generate primary Ca2+ signals, STIM proteins undergo an intricate activation reaction and rapidly translocate into junctions formed between the ER and the plasma membrane. There, STIM proteins tether and activate the highly Ca2+-selective Orai channels to mediate finely controlled Ca2+ signals and to homeostatically balance cellular Ca2+. Details are emerging on the remarkable organization within these STIM-induced junctional microdomains and the identification of new regulators and alternative target proteins for STIM.

The ubiquitously expressed Orai Ca2+ channels are gated through a unique process of intermembrane coupling with the Ca2+-sensing STIM proteins. Despite the significance of Orai1-mediated Ca2+ signals, how gating of Orai1 is triggered by STIM1 remains unknown. A widely held gating model invokes STIM1 binding directly to Orai1 pore-forming helix. Here we report that an Orai1 C-terminal STIM1-binding site, situated far from the N-terminal pore helix, alone provides the trigger that is necessary and sufficient for channel gating. We identify a critical ‘nexus' within Orai1 connecting the peripheral C-terminal STIM1-binding site to the Orai1 core helices. Mutation of the nexus transforms Orai1 into a persistently open state exactly mimicking the action of STIM1. We suggest that the Orai1 nexus transduces the STIM1-binding signal through a conformational change in the inner core helices, and that STIM1 remotely gates the Orai1 channel without the necessity for direct STIM1 contact with the pore-forming helix.

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (P o) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in P o; effective gating charge, q eff, of 2.3 ± 0.6 eo). Estimates of q eff were little changed for intracellular Ca2+ (Ca2+ i) ranging from 0.0003 to 1,024 μM. Increasing Ca2+ i from 0.03 to 1,024 μM shifted the voltage for half maximal activation (V1/2) 175 mV in the hyperpolarizing direction. V1/2 was independent of Ca2+ i for Ca2+ i ≤ 0.03 μM, indicating that the channel can be activated in the absence of Ca2+ i. Open and closed dwell-time distributions for data obtained at different Ca2+ i and voltage, but at the same P o, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of P o arose from a decrease in the mean closing rate with depolarization (q eff = −0.5 eo) and an increase in the mean opening rate (q eff = 1.8 eo), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+ i (∼0 through 1,024 μM), voltage (+80 to −80 mV), and P o (10−4 to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.

The Ca2+-dependent gating mechanism of large-conductance calcium-activated K+ (BK) channels from cultured rat skeletal muscle was examined from low (4 μM) to high (1,024 μM) intracellular concentrations of calcium (Ca2+ i) using single-channel recording. Open probability (P o) increased with increasing Ca2+ i (K 0.5 11.2 ± 0.3 μM at +30 mV, Hill coefficient of 3.5 ± 0.3), reaching a maximum of ∼0.97 for Ca2+ i ∼ 100 μM. Increasing Ca2+ i further to 1,024 μM had little additional effect on either P o or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca2+ i (>100 μM), compared with three to four open and five to seven closed states at lower Ca2+ i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca2+ i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca2+ i. Kinetic schemes drawn from Eigen's general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca2+ i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca2+ i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca2+ i. A model that assumed that the apparent Ca2+-binding steps can reach a maximum rate at high Ca2+ i could also approximate the gating from low to high Ca2+ i. The considered models can serve as working hypotheses for the gating of BK channels.