Abstract 406: Large Perinuclear Ca2+ Release Events Generated by an Interaction Between Ryanodine and IP3 Receptors in Canine Purkinje Cells
Purpose: To study the characteristics of Ca2+ release events by regions in normal canine Purkinje cells.
Methods: Cells were loaded with Fluo-4AM. Ca2+ release events (elevations of basal [Ca2+ equivalent to F/F0 3.4SD over F0) were imaged using 2D confocal microscope (16 fps). Only cells free of Ca2+ waves were analyzed using a custom IDL routine program. Subsarcolemmal region (SSL) was defined as 5μm from cell edges.
Results: 94% of events (0.0035 ±.0.0007 ev/μm2/sec, 1022 events, n=34 cells) were detected within a single frame (normal events, NE). However, a subpopulation (6.0%, 0.00022±0.00005 evμm2/sec, 150 events, n=41cells; large events, LE) lasted for several frames, showed greater spatial extent (51.0±3.9 vs NE 9.0±0.3 μm2, p<0.01) and had higher amplitude (F/F0 1.38±0.02 vs NE 1.20±0.09, p<0.01). 10μM phenylephrine increased LE event rate (n=21, P<0.01), whereas 2APB inhibited it (n=24, p<0.05). 1mM tetracaine eliminated LEs. LEs differed by subcellular region. Core LEs were 4x less frequent (0.00012±0.00004 vs 0.00052±0.00011 evm2/sec, p<0.01), had lower amplitude (F/F0 1.29±0.02 vs 1.43±0.03, p<0.01) and slower decay (p<0.05) vs SSL LEs. Spatial extent did not differ (52.2±6.0 vs 50.1±5.1μm2). While SSL LEs were scattered randomly, Core LEs were predominantly distributed longitudinally 18.2±1.6 μm from the center of the nuclei (Figure⇓) CoImmunolabeling showed that IP3R1s were located near both ends of nucleus overlapping with RyR2s.
Conclusion: In normal Purkinje cells, large Ca2+release events occur in a specific perinuclear region and are likely due to interaction between RyR2 and IP3R1. Perinuclear LE may contribute to nuclear transcription.