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Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions.

ABSTRACT: We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca²? release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.

SUBMITTER: Wong J

PROVIDER: S-EPMC3672889 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions.

Wong Joseph J Baddeley David D Bushong Eric A EA Yu Zeyun Z Ellisman Mark H MH Hoshijima Masahiko M Soeller Christian C

Biophysical journal 20130601 11

We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junc ...[more]

PMID: 23746531

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Project description:Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 +/- 8, 334 +/- 15, and 441 +/- 27 pS in symmetric 300 mM Cs(+) were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to <or=100 pS when Cs(+) was replaced with Ca(2+). The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NP(o))-voltage curve was bell shaped, with NP(o) drastically decreasing when voltage was switched from -30 to -40 mV. NP(o) increased when intracellular calcium (Ca(2+)(i)) was raised within 0.1-100 microM to abruptly diminish with higher Ca(2+)(i). Thus maximal activity occurred within the Ca(2+)(i) range found in rat cerebral artery myocytes under physiological conditions. NP(o) was reduced by ruthenium red (80 muM), increased monotonically by caffeine (0.1-5 mM) or ryanodine (0.05-5 microM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 muM), resistant to dantrolene (100 muM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.

Dataset Information

Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions.

Publications

Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions.

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