ARE CARDIAC DIHYDROPYRIDINE RECEPTORS PHYSICALLY COUPLED TO CARDIAC RYANODINE RECEPTORS?
Angela F. Dulhunty, Suzi M. Pace, Suzanne M. Curtis, Magdolena Sakowska, Philip G. Board, Marco G. Casarotto, John Curtin School of Medical Research, ACT.
Excitation-contraction (EC) coupling in cardiac muscle depends on activation of ryanodine receptor (RyR) Ca2+ release channels by Ca2+ entering through dihydropyridine receptor (DHPR) Ca2+ channels. A physical interaction between the DHPR and RyR has not been thought to occur in cardiac muscle. There is however evidence for conformational coupling between the cardiac DHPR and RyR(1,2) and peptide fragments of the cardiac and skeletal DHPR bind to the cardiac RyR and alter Ca2+ spark frequency(3,4).
To further explore the possibility that DHPRs are physically coupled to RyRs in the heart, we examined the effects of several DHPR fragments on Ca2+ release from cardiac sarcoplasmic reticulum (SR) and on cardiac RyR channels in lipid bilayers. SR vesicles were prepared from sheep heart following euthanasia by anaesthetic overdose. Cardiac peptides CC (Asp855-Pro891) and AC (Thr793-Ala812) activated RyRs at ≥10nM. Skeletal peptide CS (Glu724-Pro760), and full II-III loop (Glu666-Leu791), activated RyRs 10-100nM and inhibited at higher concentrations. These effects were independent of bilayer potential. The skeletal AS (Thr671-Leu690) activated the channels at positive and negative potentials at ≤1µM, but blocked at >1µM in a voltage-dependent manner at +40mV. RyRs were also activated by peptide AS mutants and the AS mimetic (Imperatoxin A). All fragments (except AC) enhanced Ca2+-induced Ca2+ release from SR. RyRs were not activated by the scrambled AS (ASS) or skeletal peptide (NB). We suggest that DHPRs may be physically coupled to RyRs in cardiac muscle and might directly modify Ca2+-induced Ca2+ release.
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