Biomedical subjects

M Fill

Publications and source records attributed to M Fill.

39 records · Page 3Linked to original sources

PubMed1988

Purified ryanodine receptor from rabbit skeletal muscle is the calcium-release channel of sarcoplasmic reticulum.

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS-solubilized triads using immunoaffinity chromatography. The purified receptor had a [3H]ryanodine-binding capacity (Bmax) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +/- 10 pS in 54 mM trans Ca, (c) a long-term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio PCa/PTris approximately equal to 14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (gamma max for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio PCa/PK approximately equal to 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine.

Adenosine Triphosphate↗

PubMed1986

Calcium release rate in skinned skeletal muscle fibers measured with arsenazo III.

Calcium ion release from the sarcoplasmic reticulum of single skinned (sarcolemma removed) skeletal muscle fibers was studied using the calcium-sensitive dye arsenazo III (Arz III). Isotropic absorption measurements were made differentially to reduce the effect of movement artifacts. The question of dye stoichiometry was addressed by measuring the absorption ratio at 600 and 660 nm at various times during the calcium transient. The results indicate that little change in the proportions of the various calcium-dye species occurs until at least 1 s into the release and, further, that the 1:2 calcium-dye complex is unlikely to be the dominant species present at early times. The relationship between dye concentration and the slope of the early absorption change was found to be linear for all levels of fiber loading. This suggests that the 1:1 rather than the 2:2 complex is the major species formed at early times in skinned fibers, although this conclusion is at odds with in vitro studies of Arz III in solution. Beer's law was used to convert the slope of the absorption transient measured over the first 125 ms of a release to the rate of change of the calcium-dye complex. The average rate at which the calcium-dye complex was formed was found to be 0.6 microM/ms. Two models are considered that allow calculation of a correction factor that is used to convert this value to the rate of calcium release from the sarcoplasmic reticulum. The magnitude of these correction factors was a function of the dye and intrinsic buffer concentrations as well as the stoichiometry of the calcium-dye reaction. After application of the correction factors, the average release rate in our fibers was calculated to range from 0.8 to 13.5 microM/ms.

Absorption↗

PubMedDate not supplied

Bay K 8644 increases resting Ca2+ spark frequency in ferret ventricular myocytes independent of Ca influx: contrast with caffeine and ryanodine effects.

Bay K 8644, an L-type Ca2+ channel agonist, was shown previously to increase resting sarcoplasmic reticulum (SR) Ca2+ loss and convert post-rest potentiation to decay in dog and ferret ventricular muscle. Here, the effects of Bay K 8644 on local SR Ca2+ release events (Ca2+ sparks) were measured in isolated ferret ventricular myocytes, using laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo-3. The spark frequency under control conditions was fairly constant during 20 s of rest after interruption of electrical stimulation. Bay K 8644 (100 nmol/L) increased the spark frequency by 466+/-90% of control at constant SR Ca2+ load but did not change the spatial and temporal characteristics of individual sparks. The increase in spark frequency was maintained throughout the period of rest. The increase in Ca2+ spark frequency induced by Bay K 8644 was not affected by superfusion with Ca2+-free solution (with 10 mmol/L EGTA) but was suppressed by the addition of 10 micromol/L nifedipine (which by itself did not alter resting Ca2+ spark frequency). This suggests that the effect of Bay K 8644 on Ca2+ sparks is mediated by the sarcolemmal dihydropyridine receptor but is also independent of Ca2+ influx. Low concentrations of caffeine (0.5 mmol/L) increased both the average frequency and duration of sparks. Ryanodine (50 nmol/L) increased the spark frequency and also induced long-lasting Ca2+ signals. This may indicate long-lasting openings of SR Ca2+ release channels and a lack of local SR Ca2+ depletion. In lipid bilayers, Bay K 8644 had no effect on either single-channel current amplitude or open probability of the cardiac ryanodine receptor. It is concluded that Bay K 8644 activates SR Ca2+ release at rest, independent of Ca2+ influx and perhaps through a functional linkage between the sarcolemmal dihydropyridine receptor and the SR ryanodine receptor. In contrast, caffeine and ryanodine modulate Ca2+ sparks by a direct action on the SR Ca2+ release channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗