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Single-molecule tracking of inositol trisphosphate receptors reveals different motilities and distributions.

ABSTRACT: Puffs are local Ca(2+) signals that arise by Ca(2+) liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP3Rs). The locations of puff sites observed by Ca(2+) imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP3Rs have shown that the majority of IP3Rs are freely motile. To address this discrepancy, we applied single-molecule imaging to locate and track type 1 IP3Rs tagged with a photoswitchable fluorescent protein and expressed in COS-7 cells. We found that ? 70% of the IP3R1 molecules were freely motile, undergoing random walk motility with an apparent diffusion coefficient of ? 0.095 ?m s(-1), whereas the remaining molecules were essentially immotile. A fraction of the immotile IP3Rs were organized in clusters, with dimensions (a few hundred nanometers across) comparable to those previously estimated for the IP3R clusters underlying functional puff sites. No short-term (seconds) changes in overall motility or in clustering of immotile IP3Rs were apparent following activation of IP3/Ca(2+) signaling. We conclude that stable clusters of small numbers of immotile IP3Rs may underlie local Ca(2+) release sites, whereas the more numerous motile IP3Rs appear to be functionally silent.

SUBMITTER: Smith IF

PROVIDER: S-EPMC4142249 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Single-molecule tracking of inositol trisphosphate receptors reveals different motilities and distributions.

Smith Ian F IF Swaminathan Divya D Dickinson George D GD Parker Ian I

Biophysical journal 20140801 4

Puffs are local Ca(2+) signals that arise by Ca(2+) liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP3Rs). The locations of puff sites observed by Ca(2+) imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP3Rs have shown that the majority of IP3Rs are freely motile. To address this discrepancy, we ...[more]

PMID: 25140418

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Project description:RationaleThe mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca2+ regulates gene expression, but the nature and significance of nuclear Ca2+-changes in AF are largely unknown.ObjectiveTo elucidate mechanisms by which AF alters atrial-cardiomyocyte nuclear Ca2+ ([Ca2+]Nuc) and CaMKII (Ca2+/calmodulin-dependent protein kinase-II)-related signaling.Methods and resultsAtrial cardiomyocytes were isolated from control and AF dogs (kept in AF by atrial tachypacing [600 bpm × 1 week]). [Ca2+]Nuc and cytosolic [Ca2+] ([Ca2+]Cyto) were recorded via confocal microscopy. Diastolic [Ca2+]Nuc was greater than [Ca2+]Cyto under control conditions, while resting [Ca2+]Nuc was similar to [Ca2+]Cyto; both diastolic and resting [Ca2+]Nuc increased with AF. IP3R (Inositol-trisphosphate receptor) stimulation produced larger [Ca2+]Nuc increases in AF versus control cardiomyocytes, and IP3R-blockade suppressed the AF-related [Ca2+]Nuc differences. AF upregulated nuclear protein expression of IP3R1 (IP3R-type 1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot) while decreasing the nuclear/cytosolic expression ratio for HDAC4 (histone deacetylase type-4). Isolated atrial cardiomyocytes tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca2+]Nuc changes and L-type calcium current decreases versus 1-Hz-paced cardiomyocytes; these changes were prevented by IP3R knockdown with short-interfering RNA directed against IP3R1. Nuclear/cytosolic HDAC4 expression ratio was decreased by 3-Hz pacing, while nuclear CaMKII phosphorylation was increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IP3R-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial cardiomyocytes from AF patients, nuclear IP3R1-expression was significantly increased, with decreased nuclear/nonnuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by luciferase assay) and was downregulated in AF atrial cardiomyocytes; microRNA-26a silencing reproduced AF-induced IP3R1 upregulation and nuclear diastolic Ca2+-loading.ConclusionsAF increases atrial-cardiomyocyte nucleoplasmic [Ca2+] by IP3R1-upregulation involving miR-26a, leading to enhanced IP3R1-CaMKII-HDAC4 signaling and L-type calcium current downregulation. Graphic Abstract: A graphic abstract is available for this article.

Dataset Information

Single-molecule tracking of inositol trisphosphate receptors reveals different motilities and distributions.

Publications

Single-molecule tracking of inositol trisphosphate receptors reveals different motilities and distributions.

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