Glu(106) in the Orai1 pore contributes to fast Ca(2+)-dependent inactivation and pH dependence of Ca(2+) release-activated Ca(2+) (CRAC) current

FCDI (fast Ca2+ -dependent inactivation) is a mechanism that limits Ca2+ entry through Ca2+ channels, including CRAC (Ca2+release-activated Ca2+ ) channels. This phenomenon occurs when the Ca2+ concentration rises beyond a certain level in the vicinity of the intracellular mouth of the channel pore. In CRAC channels, several regions of the pore-forming protein Orai1, and STIM1 (stromal interaction molecule 1), the sarcoplasmic/endoplasmic reticulum Ca2+ sensor that communicates the Ca2+ load of the intracellular stores to Orai1, have been shown to regulate fast Ca2+ -dependent inactivation. Although significant advances in unravelling the mechanisms of CRAC channel gating have occurred, the mechanisms regulating fast Ca2+ -dependent inactivation in this channel are not well understood. We have identified that a poremutation, E106D Orai1, changes the kinetics and voltage dependence of the ICRAC (CRAC current), and the selectivity of the Ca2+ -binding site that regulates fast Ca2+ - dependent inactivation, whereas the V102I and E190Q mutants when expressed at appropriate ratios with STIM1 have fast Ca2+ -dependent inactivation similar to that of WT (wild-type) Orai1. Unexpectedly, the E106D mutation also changes the pH dependence of ICRAC.UnlikeWTICRAC, E106D-mediated current is not inhibited at low pH, but instead the block of Na+ permeation through the E106D Orai1 pore by Ca2+ is diminished. These results suggest that Glu106 inside the CRAC channel pore is involved in co-ordinating the Ca2+ -binding site that mediates fast Ca2+ -dependent inactivation.Nathan R. Scrimgeour, David P. Wilson and Grigori Y. Rychko

Smooth muscle membrane potential modulates endothelium-dependent relaxation of rat basilar artery via myo-endothelial gap junctions

The release of endothelium-derived relaxing factors, such as nitric oxide (NO), is dependent on an increase in intracellular calcium levels ([Ca2+]i) within endothelial cells. Endothelial cell membrane potential plays a critical role in the regulation of [Ca2+]i in that calcium influx from the extracellular space is dependent on membrane hyperpolarization. In this study, the effect of inhibition of vascular smooth muscle delayed rectifier K+ (KDR) channels by 4-aminopyridine (4-AP) on endothelium-dependent relaxation of rat basilar artery to acetylcholine (ACh) was assessed. ACh-evoked endothelium-dependent relaxations were inhibited by N-(Ω)-nitro-l-arginine (l-NNA) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), confirming a role for NO and guanylyl cyclase. 4-AP (300 μm) also suppressed ACh-induced relaxation, with the maximal response reduced from ≈92 to ≈33 % (n = 11; P < 0.01). However, relaxations in response to exogenous NO, applied in the form of authentic NO, sodium nitroprusside or diethylamineNONOate (DEANONOate), were not affected by 4-AP treatment (n = 3-11). These data are not consistent with the view that 4-AP-sensitive KDR channels are mediators of vascular hyperpolarization and relaxation in response to endothelium-derived NO. Inhibition of ACh-evoked relaxation by 4-AP was reversed by pinacidil (0.5-1 μm; n = 5) or 18β-glycyrrhetinic acid (18βGA; 5 μm; n = 5), indicating that depolarization and electrical coupling of the smooth muscle to the endothelium were involved. 4-AP caused depolarization of both endothelial and vascular smooth muscle cells of isolated segments of basilar artery (mean change 11 ± 1 and 9 ± 2 mV, respectively; n = 15). Significantly, 18βGA almost completely prevented the depolarization of endothelial cells (n = 6), but not smooth muscle cells (n = 6) by 4-AP. ACh-induced hyperpolarization of endothelium and smooth muscle cells was also reduced by 4-AP, but this inhibition was not observed in the combined presence of 4-AP and 18βGA. These data indicate that 4-AP can induce an indirect inhibition of endothelium-dependent relaxation in the rat basilar artery by electrical coupling of smooth muscle membrane depolarization to the endothelium via myo-endothelial gap junctions

Identification of key amino acid residues responsible for internal and external pH sensitivity of Orai1/STIM1 channels

Changes of intracellular and extracellular pH are involved in a variety of physiological and pathological processes, in which regulation of the Ca(2+) release activated Ca(2+) channel (I(CRAC)) by pH has been implicated. Ca(2+) entry mediated by I(CRAC) has been shown to be regulated by acidic or alkaline pH. Whereas several amino acid residues have been shown to contribute to extracellular pH (pH(o)) sensitivity, the molecular mechanism for intracellular pH (pH(i)) sensitivity of Orai1/STIM1 is not fully understood. By investigating a series of mutations, we find that the previously identified residue E106 is responsible for pH(o) sensitivity when Ca(2+) is the charge carrier. Unexpectedly, we identify that the residue E190 is responsible for pH(o) sensitivity when Na(+) is the charge carrier. Furthermore, the intracellular mutant H155F markedly diminishes the response to acidic and alkaline pH(i), suggesting that H155 is responsible for pH(i) sensitivity of Orai1/STIM1. Our results indicate that, whereas H155 is the intracellular pH sensor of Orai1/STIM1, the molecular mechanism of external pH sensitivity varies depending on the permeant cations. As changes of pH are involved in various physiological/pathological functions, Orai/STIM channels may be an important mediator for various physiological and pathological processes associated with acidosis and alkalinization

Status and initial physics performance studies of the MPD experiment at NICA

The Nuclotron-based Ion Collider fAcility (NICA) is under construction at the Joint Institute for Nuclear Research (JINR), with commissioning of the facility expected in late 2022. The Multi-Purpose Detector (MPD) has been designed to operate at NICA and its components are currently in production. The detector is expected to be ready for data taking with the first beams from NICA. This document provides an overview of the landscape of the investigation of the QCD phase diagram in the region of maximum baryonic density, where NICA and MPD will be able to provide significant and unique input. It also provides a detailed description of the MPD set-up, including its various subsystems as well as its support and computing infrastructures. Selected performance studies for particular physics measurements at MPD are presented and discussed in the context of existing data and theoretical expectations