Ligand sensitivity of type-1 inositol 1,4,5-trisphosphate receptor is enhanced by the D2594K mutation

Abstract

Inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) are homologous cation channels that mediate release of Ca²⁺ from the endoplasmic/sarcoplasmic reticulum (ER/SR) and thereby are involved in many physiological processes. In previous studies, we determined that when the D2594 residue, located at or near the gate of the IP₃R type 1, was replaced by lysine (D2594K), a gain of function was obtained. This mutant phenotype was characterized by increased IP₃ sensitivity. We hypothesized the IP₃R1-D2594 determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states. To test this possibility, the relationship between the D2594 site and IP₃R1 regulation by IP₃, cytosolic, and luminal Ca²⁺ was determined at the cellular, subcellular, and single-channel levels using fluorescence Ca²⁺ imaging and single-channel reconstitution. We found that in cells, D2594K mutation enhances the IP₃ ligand sensitivity. Single-channel IP₃R1 studies revealed that the conductance of IP₃R1-WT and -D2594K channels is similar. However, IP₃R1-D2594K channels exhibit higher IP₃ sensitivity, with substantially greater efficacy. In addition, like its wild type (WT) counterpart, IP₃R1-D2594K showed a bell-shape cytosolic Ca²⁺-dependency, but D2594K had greater activity at each tested cytosolic free Ca²⁺ concentration. The IP₃R1-D2594K also had altered luminal Ca²⁺ sensitivity. Unlike IP₃R1-WT, D2594K channel activity did not decrease at low luminal Ca²⁺ levels. Taken together, our functional studies indicate that the substitution of a negatively charged residue by a positive one at the channels' pore cytosolic exit affects the channel's gating behavior thereby explaining the enhanced ligand-channel's sensitivity.

Keywords: Ca2+ puffs; Gain of function; IP3; Inositol 1,4,5-trisphosphate receptor; Single channels.