title: Quantifying SOCE fluorescence measurements in mammalian muscle fibres. The effects of ryanodine and osmotic shocks.
abstract: We have quantified Ca(2+) entry through store operated calcium channels in mice muscle fibres, measuring the rates of change of myoplasmic [Ca(2+)], d[Ca(2+)](myo)/dt, and of Ca(2+) removal, d[Ca(2+)](Removal)/dt, turning store operated calcium entry (SOCE) ON, and OFF, by switching on or off external Ca(2+). In depleted fibres, poisoned with 10 μM cyclopiazonic acid SOCE influx was about 3 μM/s. Ryanodine (50 μM) caused a robust, nifedipine (50 μM) independent, increase in SOCE activation to 8.6 μM/s. Decreasing medium osmolarity from 300 to 220 mOsm/L, decreased SOCE to 0.9 μM/s, while increasing osmolarity from 220 to 400 mOsm/L potentiated SOCE to 43.6 μM/s. Ryanodine inhibited the effects of hypotonicity. Experiments using 2-aminoethoxydiphenyl borate, nifedipine, or Mn(2+) quenching, strongly suggest that the increased [Ca(2+)](myo) by ryanodine or hypertonic shock is mediated by potentiated SOCE activation. The Ca(2+) response decay, quantified by d[Ca(2+)](Removal)/dt, indicates a robust residual Ca(2+) removal mechanism in sarco-endoplasmic reticulum calcium ATPase poisoned fibres. SOCE high sensitivity to osmotic shocks, or to ryanodine receptor (RyR) binding, suggests its high dependency on the structural relationship between its molecular constituents, Orai1 and stromal interaction molecule and the sarcoplasmic reticulum and plasma membranes, in the triadic junctional region, where RyRs, are conspicuously present. This study demonstrates that SOCE machinery is highly sensitive to structural changes caused by binding of an agonist to its receptor or by imposed osmotical volume changes.
authors.affiliation: Laboratorio de Fisiología Celular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela, bolanosp@gmail.com.
authors.forename: Pura
authors.initials: P
authors.lastname: Bolaños
authors.forename: Alis
authors.initials: A
authors.lastname: Guillen
authors.forename: Adriana
authors.initials: A
authors.lastname: Gámez
authors.forename: Carlo
authors.initials: C
authors.lastname: Caputo
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D005453:Fluorescence; D007211:Indoles / Q000494:pharmacology; D008297:Male; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism*; D009997:Osmotic Pressure; D012433:Ryanodine / Q000494:pharmacology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D007211:Indoles; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002118:Calcium; C000543:cyclopiazonic acid