Photochemical gating of intracellular Ca2+ release channels.
Ontology highlight
ABSTRACT:
SUBMITTER: Ni J
PROVIDER: S-EPMC2536572 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The ubiquitous inositol 1,4,5-trisphosphate receptor (InsP(3)R) intracellular Ca(2+) release channel is engaged by thousands of plasma membrane receptors to generate Ca(2+) signals in all cells. Understanding how complex Ca(2+) signals are generated has been hindered by a lack of information on the kinetic responses of the channel to its primary ligands, InsP(3) and Ca(2+), which activate and inhibit channel gating. Here, we describe the kinetic responses of single InsP(3)R channels in native endoplasmic reticulum membrane to rapid ligand concentration changes with millisecond resolution, using a new patch-clamp configuration. The kinetics of channel activation and deactivation showed novel Ca(2+) regulation and unexpected ligand cooperativity. The kinetics of Ca(2+)-mediated channel inhibition showed the single-channel bases for fundamental Ca(2+) release events and Ca(2+) release refractory periods. These results provide new insights into the channel regulatory mechanisms that contribute to complex spatial and temporal features of intracellular Ca(2+) signals.
| S-EPMC2247393 | biostudies-literature
Project description:Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of ∼2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.
| S-EPMC3156176 | biostudies-literature
Project description:Store-operated Ca(2+) entry (SOCE) due to activation of Ca(2+) release-activated Ca(2+) (CRAC) channels leads to sustained elevation of cytoplasmic Ca(2+) and activation of lymphocytes. CRAC channels consisting of four pore-forming Orai1 subunits are activated by STIM1, an endoplasmic reticulum Ca(2+) sensor that senses intracellular store depletion and migrates to plasma membrane proximal regions to mediate SOCE. One of the fundamental properties of CRAC channels is their Ca(2+)-dependent fast inactivation. To identify the domains of Orai1 involved in fast inactivation, we have mutated residues in the Orai1 intracellular loop linking transmembrane segment II to III. Mutation of four residues, V(151)SNV(154), at the center of the loop (MutA) abrogated fast inactivation, leading to increased SOCE as well as higher CRAC currents. Point mutation analysis identified five key amino acids, N(153)VHNL(157), that increased SOCE in Orai1 null murine embryonic fibroblasts. Expression or direct application of a peptide comprising the entire intracellular loop or the sequence N(153)VHNL(157) blocked CRAC currents from both wild type (WT) and MutA Orai1. A peptide incorporating the MutA mutations had no blocking effect. Concatenated Orai1 constructs with four MutA monomers exhibited high CRAC currents lacking fast inactivation. Reintroduction of a single WT monomer (MutA-MutA-MutA-WT) was sufficient to fully restore fast inactivation, suggesting that only a single intracellular loop can block the channel. These data suggest that the intracellular loop of Orai1 acts as an inactivation particle, which is stabilized in the ion permeation pathway by the N(153)VHNL(157) residues. These results along with recent reports support a model in which the N terminus and the selectivity filter of Orai1 as well as STIM1 act in concert to regulate the movement of the intracellular loop and evoke fast inactivation.
| S-EPMC2836109 | biostudies-literature
Project description:A large (565 kDa) junctional sarcoplasmic reticulum (SR) protein, the ryanodine receptor (RYR), may play both a structural and a functional role in the mechanism of skeletal muscle excitation-contraction coupling. Recently, the primary amino acid sequence of the RYR has been elucidated. In this paper, we introduce an immunological approach to examine the functional (electrophysiological) properties of the RYR when it is incorporated into planar lipid bilayers. The effects of two polyclonal antibodies against the SR junctional face membrane (JFM) and the RYR (anti-JFM and anti-RYR) were tested on the single-channel gating properties of the RYR SR Ca2(+)-release channel. Junctional SR vesicles were fused into planar lipid bilayers in solutions containing caesium salts. Solutions were designed to minimize the background conductances of the SR K+ and Cl- channels. Three actions of the anti-JFM antibody were distinguished on the basis of single-channel gating and conductance. The anti-RYR antibody had a single action, a simultaneous decrease in single-channel open probability (Po) and conductance. Both antibodies appear to alter single-channel gating by disrupting the Ca2(+)-activation mechanism of the channel. Anti-RYR-antibody-induced gating abnormalities were reversed by ATP, although the ATP-re-activated channels had altered gating kinetics. Two antigenic regions, recognizing the anti-RYR antibody, in the C-terminal end of the RYR primary amino acid sequence contain or are closely associated with putative ligand (Ca2+ and ATP)-binding sites identified previously. Our results demonstrate (1) that the antibodies induced abnormal gating (decreased open probability and stabilization of subconducting states) of SR release channels, and (2) that abnormal gating is not associated with physical obstruction or alteration of the conduction pathway. Thus antibodies directed at specific regions of the RYR (e.g. putative ligand-binding sites) can be used as effective probes with which to study the structural and functional properties of the SR Ca2(+)-release channel gating at the single-channel level.
| S-EPMC1149866 | biostudies-other
Project description:Small-conductance Ca2+-activated K+ (SK) channels are widely expressed in neuronal tissues where they underlie post-spike hyperpolarizations, regulate spike-frequency adaptation, and shape synaptic responses. SK channels constitutively interact with calmodulin (CaM), which serves as Ca2+ sensor, and with protein kinase CK2 and protein phosphatase 2A, which modulate their Ca2+ gating. By recording coupled activities of Ca2+ and SK2 channels, we showed that SK2 channels can be inhibited by neurotransmitters independently of changes in the activity of the priming Ca2+ channels. This inhibition involvesSK2-associated CK2 and results from a 3-fold reduction in the Ca2+ sensitivity of channel gating. CK2phosphorylated SK2-bound CaM but not KCNQ2-bound CaM, thereby selectively regulating SK2 channels. We extended these observations to sensory neurons by showing that noradrenaline inhibits SK current and increases neuronal excitability in aCK2-dependent fashion. Hence, neurotransmitter-initiated signaling cascades can dynamically regulate Ca2+ sensitivity of SK channels and directly influence somatic excitability.
| S-EPMC2651825 | biostudies-literature
Project description:The Ca2+-dependent gating mechanism of large-conductance calcium-activated K+ (BK) channels from cultured rat skeletal muscle was examined from low (4 microM) to high (1,024 microM) intracellular concentrations of calcium (Ca2+i) using single-channel recording. Open probability (Po) increased with increasing Ca2+i (K0. 5 11.2 +/- 0.3 microM at +30 mV, Hill coefficient of 3.5 +/- 0.3), reaching a maximum of approximately 0.97 for Ca2+i approximately 100 microM. Increasing Ca2+i further to 1,024 microM had little additional effect on either Po 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 microM), 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.
| S-EPMC2229641 | biostudies-literature
Project description:Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.
| S-EPMC2234499 | biostudies-literature
Project description:Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation-contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.
| S-EPMC2064307 | biostudies-literature
Project description:Ca(2+) influx by store-operated Ca(2+) influx channels (SOCs) mediates many cellular functions regulated by Ca(2+), and excessive SOC-mediated Ca(2+) influx is cytotoxic and associated with disease. One form of SOC is the CRAC current that is mediated by Orai channels activated by STIM1. A fundamental property of the native CRAC and of the Orais is fast Ca(2+)-dependent inactivation, which limits Ca(2+) influx to guard against cellular damage. The molecular mechanism of this essential regulatory mechanism is unknown. We report here the fast Ca(2+)-dependent inactivation is mediated by three conserved glutamates in the C termini (CT) of Orai2 and Orai3, which show prominent fast Ca(2+)-dependent inactivation compared with Orai1. Transfer of the CT between the Orais transfers both the extent of channel opening and the mode of fast Ca(2+)-dependent inactivation. Fast Ca(2+)-dependent inactivation of the Orais also requires a domain of STIM1; fragments of STIM1 that efficiently open Orai channels do not evoke fast inactivation unless they include an anionic sequence that is C-terminal to the STIM1-Orai activating region (SOAR). Our studies suggest that Orai CT are necessary and sufficient to control pore opening and uncover the molecular mechanism of fast Ca(2+)-dependent inactivation that has implications for Ca(2+) influx by SOC in physiological and pathological states.
| S-EPMC2732841 | biostudies-literature
Project description:RationaleL-Type (Cav1.2) Ca(2+) channels are critical regulators of muscle and neural function. Although Cav1.2 channel activity varies regionally, little is known about the mechanisms underlying this heterogeneity.ObjectiveTo test the hypothesis that Cav1.2 channels can gate coordinately.Methods and resultsWe used optical and electrophysiological approaches to record Cav1.2 channel activity in cardiac, smooth muscle, and tsA-201 cells expressing Cav1.2 channels. Consistent with our hypothesis, we found that small clusters of Cav1.2 channels can open and close in tandem. Fluorescence resonance energy transfer and electrophysiological studies suggest that this coupling of Cav1.2 channels involves transient interactions between neighboring channels via their C termini. The frequency of coupled gating events increases in hypertensive smooth muscle and in cells expressing a mutant Cav1.2 channel that causes arrhythmias and autism in humans with Timothy syndrome (LQT8).ConclusionsCoupled gating of Cav1.2 channels may represent a novel mechanism for the regulation of Ca(2+) influx and excitability in neurons, cardiac, and arterial smooth muscle under physiological and pathological conditions.
| S-EPMC2834417 | biostudies-literature