IP3 R1-Mediated Local Ca2+ Release Events are Enhanced in the Gain-of-Function D2594K Mutant Channel

Abstract

Quantal Ca2+ release through the inositol 1,4,5-trisphosphate receptor (IP3R) is a well-defined and ubiquitous phenomenon. However, its molecular origin remains poorly understood. Our general assumption is that this process results from variations of the channel's IP3 affinity as a function of the intra-pore [Mg2+]/[Ca2+] ratio. This ratio may vary during Ca2+ release and could be sensed by a residue (D2594) located in the inner portion of the channel's permeation pore. The hypothesis tested in this work is that the gain-of-function behavior shown by the IP3R when the D2594 residue is replaced by the basic residue K, is explained by increased IP3 affinity of the channel. If so, we predicted this mutation would modify the biophysical attributes of the resulting local Ca2+ release events (puffs) resulting in larger, brighter, and/or longer puffs. To test this, Ca2+ puffs were recorded with real-time, fast Ca2+-imaging, confocal microscopy after perfusing the purinergic ligand ATP (30 & 50 nM) and puff biophysical attributes analyzed. Experiments were conducted in intact HEK cells expressing either WT or D2594K mutant IP3R. Our results indicate that D2594K transfected intact HEK cells exhibited Ca2+ puffs with no significant differences in duration, but more than 6-fold larger amplitude (WT 0.88 ± 0.24 deltaF/F0; D2594 6.1 ± 0.24 deltaF/F0) than HEK cells expressing the WT channel. In addition, D2594K-expressing cells showed more than 1.5-fold higher Ca2+ puff frequency at every tested [ATP] (WT: 2.8 & 3.3 puffs/cell⋅min at 30 & 50 nM ATP, respectively. D2594: 4.8 & 5.3 puffs/cell⋅min at 30 & 50 nM ATP, respectively). These results are consistent with our hypothesis that quantal IP3 R-mediated Ca2+ release is controlled by the D2594 residue which allosterically affects the channel's affinity for IP3.