Ca 2+ entry through CRAC channels causes fast Ca 2+ -dependent inactivation (CDI). Previous mutagenesis studies have implicated Orai1 residues W76 and Y80 in CDI through their role in binding calmodulin (CaM), in agreement with the crystal structure of Ca 2+ –CaM bound to an Orai1 N-terminal peptide. However, a subsequent Drosophila melanogaster Orai crystal structure raises concerns about this model, as the side chains of W76 and Y80 are predicted to face the pore lumen and create a steric clash between bound CaM and other Orai1 pore helices. We further tested the functional role of CaM using several dominant-negative CaM mutants, none of which affected CDI. Given this evidence against a role for pretethered CaM, we altered side-chain volume and charge at the Y80 and W76 positions to better understand their roles in CDI. Small side chain volume had different effects at the two positions: it accelerated CDI at position Y80 but reduced the extent of CDI at position W76. Positive charges at Y80 and W76 permitted partial CDI with accelerated kinetics, whereas introducing negative charge at any of five consecutive pore-lining residues (W76, Y80, R83, K87, or R91) completely eliminated CDI. Noise analysis of Orai1 Y80E and Y80K currents indicated that reductions in CDI for these mutations could not be accounted for by changes in unitary current or open probability. The sensitivity of CDI to negative charge introduced into the pore suggested a possible role for anion binding in the pore. However, although Cl − modulated the kinetics and extent of CDI, we found no evidence that CDI requires any single diffusible cytosolic anion. Together, our results argue against a CDI mechanism involving CaM binding to W76 and Y80, and instead support a model in which Orai1 residues Y80 and W76 enable conformational changes within the pore, leading to CRAC channel inactivation.

The inactivation domain of STIM1 (ID STIM : amino acids 470–491) has been described as necessary for Ca 2+ -dependent inactivation (CDI) of Ca 2+ release–activated Ca 2+ (CRAC) channels, but its mechanism of action is unknown. Here we identify acidic residues within ID STIM that control the extent of CDI and examine functional interactions of ID STIM with Orai1 pore residues W76 and Y80. Alanine scanning revealed three ID STIM residues (D476/D478/D479) that are critical for generating full CDI. Disabling ID STIM by a triple alanine substitution for these three residues (“STIM1 3A”) or by truncation of the entire domain (STIM1 1–469 ) reduced CDI to the same residual level observed for the Orai1 pore mutant W76A (approximately one third of the extent seen with full-length STIM1). Results of noise analysis showed that STIM1 1–469 and Orai1 W76A mutants do not reduce channel open probability or unitary Ca 2+ conductance, factors that determine local Ca 2+ accumulation, suggesting that they diminish CDI instead by inhibiting the CDI gating mechanism. We tested for functional coupling between ID STIM and the Orai1 pore by double-mutant cycle analysis. The effects on CDI of mutations disabling ID STIM or W76 were not additive, demonstrating that ID STIM and W76 are strongly coupled and act in concert to generate full-strength CDI. Interestingly, disabling ID STIM and W76 separately gave opposite results in Orai1 Y80A channels: channels with W76 but lacking ID STIM generated approximately two thirds of the WT extent of CDI but those with ID STIM but lacking W76 completely failed to inactivate. Together, our results suggest that Y80 alone is sufficient to generate residual CDI, but acts as a barrier to full CDI. Although ID STIM is not required as a Ca 2+ sensor for CDI, it acts in concert with W76 to progress beyond the residual inactivated state and enable CRAC channels to reach the full extent of inactivation.

Most voltage-gated K + currents are relatively insensitive to extracellular Na + (Na + o ), but Na + o potently inhibits outward human ether-a-go-go–related gene (HERG)–encoded K + channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429–30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na + (Na + i ) and extracellular Ba 2+ (Ba 2+ o ), to define a site where Na + o interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na + o was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K + selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na + o . Quantitatively similar effects of Na + i to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na + o sensitivity, this result suggested that Na + o and Na + i act at different sites. Extracellular Ba 2+ (Ba 2+ o ) blocks K + channel pores, and thereby serves as a useful probe of K + channel structure. HERG channel inactivation promotes relief of Ba 2+ block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265–278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na + o action. A remote allosteric model predicts that Na + o will speed relief of Ba 2+ o block by promoting inactivation. Instead, Na + o slowed Ba 2+ egress and Ba 2+ relieved Na + o inhibition, consistent with Na + o binding to an outer pore site. The apparent affinities of the outer pore for Na + o and K + o as measured by slowing of Ba 2+ egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na + o inhibition. Na + o inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.

Human ether-à-go-go –related gene ( HERG ) encoded K + channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca 2+ concentrations and physiological external K + . Elevation of external Ca 2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K + current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 ± 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca 2+ . Neither Ca 2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca 2+ on Human ether-à-go-go –related gene current amplitude and kinetics. The differential effects of extracellular Ca 2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca 2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca 2+ binding to this site is ineffective in modifying the inactivation gating machinery.