Activation of calcium channels in sarcoplasmic reticulum from frog muscle by nanomolar concentrations of ryanodine

Abstract

Sarcoplasmic reticulum vesicles isolated from fast-twitch frog skeletal muscle presented two classes of binding sites for ryanodine, one of high affinity (Kd1 = 1.7 nM, Bmax1 = 3.3 pmol per mg) and a second class with lower affinity (Kd2 = 90 nM, Bmax2 = 7.0 pmol per milligram). The calcium channels present in the sarcoplasmic reticulum membranes were studied in vesicles fused into lipid bilayers. Low concentrations of ryanodine (5 to 10 nM) activated a large conductance calcium channel after a short delay (5 to 10 min). The activation, which could be elicited from conditions of high or low fractional open time, was characterized by an increase in channel fractional open time without a change in conductance. The open and closed dwell time distributions were fitted with the sum of two exponentials in the range of 4 to 800 ms. The activating effect of ryanodine was due to an increase of both open time constants and a concomitant decrease in the closed time constants. Under conditions of low fractional open time (less than 0.1), the time spent in long closed periods (greater than 800 ms) between bursts was not affected by ryanodine. Higher concentrations of ryanodine (250 nM) locked the channel in a lower conductance level (approximately 40%) with a fractional open time near unity. These results suggest that the activating effects of nanomolar concentrations of ryanodine may arise from drug binding to high affinity sites. The expression of the lower conductance state obtained with higher concentrations of ryanodine may be associated with the low affinity binding sites observed in frog sarcoplasmic reticulum.

Calcium dependence of ryanodine‐sensitive calcium channels from brain cortex endoplasmic reticulum

“…Binding of [3H]ryanodine was assayed as described previously [16], except that in some experiments 10 [aM [Ca 2+] was present in the incubation solution (adjusted with 2 mM HEDTA and 1.56 mM CaClz). Binding of [3H]saxitoxin ([3H]STX) was assayed using a modification of a previous protocol [17]; briefly, membranes at 0.5 mg of protein per ml were incubated for 1 h at 22°C in 0.3 ml of a solution containing 120 mM choline chloride, 2.5 mM KCI, 1.…”

Structural and functional aspects of calcium homeostasis in eukaryotic cells

“…There is also evidence of voltage dependence on the opening probability of the Ca2 ' -release channel in the lipid bilayers [238, 2571. Ryanodine at concentrations close to the Kd of high-affinity binding (5 -50 nM) increases the opening probability of the channel during its bursting periods [258], while at micromolar concentrations ryanodine locks the channel in a low-conductance state with an opening probability near unity [258].…”

“…There is also evidence of voltage dependence on the opening probability of the Ca2 ' -release channel in the lipid bilayers [238, 2571. Ryanodine at concentrations close to the Kd of high-affinity binding (5 -50 nM) increases the opening probability of the channel during its bursting periods [258], while at micromolar concentrations ryanodine locks the channel in a low-conductance state with an opening probability near unity [258].Using ryanodine, the Ca2 '-release channel has been purified from both skeletal and cardiac muscle [259 -2621. The purified ryanodine receptor has been shown to function as a Ca2+-release channel when reconstituted into planar lipid bilayers with biophysical properties similar, but not identical, 609 tion of the triad junction of the skeletal muscle is shown in Fig.…”

Sarcoplasmic reticulum release channels from frog skeletal muscle display two types of calcium dependence

“…They are activated by millimolar ATP and micromolar Ca'+, and are blocked by millimolar MgZ' and micromolar Ruthenium red added to the cytosolic side of the channel [9-l 51. Nanomolar ryanodine increases fractional open time (P,), with no change in channel conductance [ 16,171, whereas micromolar ryanodine locks the channel in a low conductance state with P, close to unity [l&18].…”