Thirty-eighth Annual Meeting March 6-10, 1994 New Orleans Convention Center New Orleans, Louisiana. Tuesday Symposia and Posters, Part III

1994 Biophysical Journal  
To estimate the amount of Ca released from the intracellular Ca pool with each opening of an inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel, we measured permeation properties of this channel after it was incorporated into planar bilayers. Channel currents were recorded after addition of 2 AM InsP3 and 0.5 mM ATP to the cytoplasmic (cis) side and with Ba or Ca on the lumenal (trans) side as the current carrier. The following results were obtained: (1) With a transmembrane voltage of 0 mV,
more » ... the unitary current was 1.6 times smaller with Ca than with Ba as the current carrier. With 55 mM divalent cation trans/l 10 mM Tris cis, the single channel current was 2.25 + 0.07 pA (n=8) with Ba and 1.4 + 0.04 pA (n=6) with Ca. (2) When InsP3gated channels were measured at different trans Ba and Ca concentrations, the half-maximal unitary current was obtained at 10 mM for each of these cations. (3) When 110 mM potassium was used in the cis chamber instead of Tris, the unitary current with Ba as the current carrier was 1.75 times larger; the current was 3.95 + 0.12 pA (n =5). From these results the size of the unitary current under physiological conditions (1 mM free Ca in the lumen of the reticulum/l 10 mM K in the cytosol) was estimated to be 0.26 pA. With this current and the mean open time of the channel observed in our experiments of 5 ms, approximately 4000 Ca ions will be released into the cytosol each time a channel opens. The estimated amount of Ca released at each opening of the ryanodine receptor is 10-100 times larger. Thus, the existence of two types of intracellular Ca channels may account for the wide range in the observed rates of change of cytosolic Ca. The apparent concentrations of intra sarcoplasmic reticulum (SR) free Ca2+ were estimated in fura-2AM-loaded, saponin-skinned cultured aortic smooth muscle cells (A7rS). Images of intracellular fura-2 fluorescence elicited by illumination at 360 nm (isosbestic point) revealed that after cell skinning cytosolic fura-2 was washed from continuously superfused preparations and only fura-2 that was trapped, primarily, in SR remained. Under these conditions, SR Ca2'was estimated to range from 1200-1600 nM and remained stable during superfusion with ATP-containing intracellular solution for up to 20 min. When cells were exposed to I eM IP3, a biphasic response was observed. There was an initial transient rise in apparent SR Ca2" which was followed by rapid decline during which 80-90% of SR Ca2" was released. Intra-SR Ca2'transients ranged from 200-600 nM in amplitude and had durations of -1min. In contrast, gradual decreases or increases in SR Ca2' were observed in skinned cells superfused with intracellular solutions containing either [Ca2+I=0 or 2000 nM, respectively. When cells were exposed to 51SM Br-A23187, there was a rapid monotonic decline in SR Ca2' until SR depletion was complete. Elevation of SR Ca2'were not observed in the presence of Br-A23 187. These results suggest that, in contrast to Br-A23 187, IP3 may elicit a rise in intra-SR free Ca2', perhaps as a consequence of decreased buffering by SR Ca2+ binding proteins. In this way, agonist evoked SR Ca2' release which is mediated by IP3 should be facilitated.
doi:10.1016/s0006-3495(94)80820-3 fatcat:yoyn5cbiyfgjti7raff4zvm5ly