Unravelling calcium-release channel gating: clues from a 'hot' disease.
Ontology highlight
ABSTRACT:
SUBMITTER: McCarthy TV
PROVIDER: S-EPMC1224198 | biostudies-other | 2004 Jun
REPOSITORIES: biostudies-other
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Project description:Glucocorticoids (GCs) are rapidly released in response to stress and play an important role in the physiological adjustments to re-establish homeostasis. The mode of action of GCs for stress coping is mediated largely by the steroid binding to the glucocorticoid receptor (GR), a ligand-bound transcription factor, and modulating the expression of target genes. However, GCs also exert rapid actions that are independent of transcriptional regulation by modulating second messenger signaling. However, a membrane-specific protein that transduces rapid GCs signal is yet to be characterized. Here, using freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and fura2 fluorescence microscopy, we report that stressed levels of cortisol rapidly stimulate the rise in cytosolic free calcium ([Ca2+]i). Pharmacological manipulations using specific extra- and intra-cellular calcium chelators, plasma membrane and endoplasmic reticulum channel blockers and receptors, indicated extracellular Ca2+ entry is required for the cortisol-mediated rise in ([Ca2+]i). Particularly, the calcium release-activated calcium (CRAC) channel gating appears to be a key target for the rapid action of cortisol in the ([Ca2+]i) rise in trout hepatocytes. To test this further, we carried out in silico molecular docking studies using the Drosophila CRAC channel modulator 1 (ORAI1) protein, the pore forming subunit of CRAC channel that is highly conserved. The result predicts a putative binding site on CRAC for cortisol to modulate channel gating, suggesting a direct, as well as an indirect regulation (by other membrane receptors) of CRAC channel gating by cortisol. Altogether, CRAC channel may be a novel cortisol-gated Ca2+ channel transducing rapid nongenomic signalling in hepatocytes during acute stress.
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Project description:A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Ca(v)1.3 L-type Ca(2+) channels, in particular the voltage dependence of activation (V(0.5)) and Ca(2+) dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Ca(v)1.3 (Ca(v)1.3(L)), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCa(v)1.3(scg)). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCa(v)1.3(scg) with a rat cDNA isolated from rat pancreas (rCa(v)1.3(L)). When expressed in tsA-201 cells under identical experimental conditions rCa(v)1.3(L) exhibited Ca(2+) current properties indistinguishable from human and mouse Ca(v)1.3(L), compatible with the presence of a functional CTM. In contrast, rCa(v)1.3(scg) showed gating properties similar to human short splice variants lacking a CTM. rCa(v)1.3(scg) differs from rCa(v)1.3(L) at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-terminal polymethionine stretch with two additional lysines. Two aa (S244, A2075) in rCa(v)1.3(scg) explained most of the functional differences to rCa(v)1.3(L). Their mutation to the corresponding residues in rCa(v)1.3(L) (G244, V2075) revealed that both contributed to the more negative V 0.5, but caused opposite effects on CDI. A2075 (located within a region forming the CTM) additionally permitted higher channel open probability. The cooperative action in the double-mutant restored gating properties similar to rCa(v)1.3(L). We found no evidence for transcripts containing one of the single rCa(v)1.3(scg) mutations in rat superior cervical ganglion preparations. However, the rCa(v)1.3(scg) variant provided interesting insight into the structural machinery involved in Ca(v)1.3 gating.
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Project description:Store-operated calcium (Ca(2+)) entry is the process by which molecules located on the endo/sarcoplasmic reticulum (ER/SR) respond to decreased luminal Ca(2+) levels by signaling Ca(2+) release activated Ca(2+) channels (CRAC) channels to open on the plasma membrane (PM). This activation of PM CRAC channels provides a sustained cytosolic Ca(2+) elevation associated with myriad physiological processes. The identities of the molecules which mediate SOCE include stromal interaction molecules (STIMs), functioning as the ER/SR luminal Ca(2+) sensors, and Orai proteins, forming the PM CRAC channels. This review examines the current available high-resolution structural information on these CRAC molecular components with particular focus on the solution structures of the luminal STIM Ca(2+) sensing domains, the crystal structures of cytosolic STIM fragments, a closed Orai hexameric crystal structure and a structure of an Orai1 N-terminal fragment in complex with calmodulin. The accessible structural data are discussed in terms of potential mechanisms of action and cohesiveness with functional observations.
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