Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes

Abstract

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 ≤100 pS when Cs+was replaced with Ca2+. The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NPo)-voltage curve was bell shaped, with NPodrastically decreasing when voltage was switched from −30 to −40 mV. NPoincreased when intracellular calcium (Ca2+i) was raised within 0.1–100 μM to abruptly diminish with higher Ca2+i. Thus maximal activity occurred within the Ca2+irange found in rat cerebral artery myocytes under physiological conditions. NPowas reduced by ruthenium red (80 μM), increased monotonically by caffeine (0.1–5 mM) or ryanodine (0.05–5 μM), 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 μM), resistant to dantrolene (100 μM), 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.