Calcium regulation of inositol 1,4,5-trisphosphate receptors

Abstract

Ca 2+ exerts both a stimulatory and inhibitory effect on type-I IP 3R channel activity. However, the structural determinants of Ca 2+ sensing in IP 3Rs are not fully understood. Previous studies by others have identified eight domains of the type-I IP 3R that bind 45Ca 2+ when expressed as GST-fusion proteins. We have mutated six highly conserved acidic residues within the second of these domains (aa378–450) in the full-length IP 3R and measured the Ca 2+regulation of IP 3-mediated Ca 2+ release in COS-7 cells. 45Ca 2+flux assays measured with a maximal [IP 3] (1 μM) indicate that one of the mutants retained a Ca 2+ sensitivity that was not significantly different from control (E411Q), three of the mutants show an enhanced Ca 2+ inhibition (D426N, E428Q and E439Q) and two of the mutants were relatively insensitive to Ca 2+ inhibition (D442N and D444N). IP 3 dose–response relationships indicated that the sensitivity to Ca 2+ inhibition and affinity for IP 3 were correlated for three of the constructs. Other mutants with enhanced IP 3 sensitivity (e.g. R441Q and a type-II/I IP 3R chimera) were also less sensitive to Ca 2+ inhibition. We conclude that the acidic residues within the aa378–450 segment are unlikely to represent a single functional Ca 2+ binding domain and do not contribute to Ca 2+ activation of the receptor. The different effects of the mutations may be related to their location within two clusters of acidic residues identified in the crystal structure of the ligand-binding domain [I. Bosanac, J.R. Alattia, T.K. Mal, et al., Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696–700]. The data support the view that all IP 3R isoforms may display a range of Ca 2+ sensitivities that are determined by multiple sites within the protein and markedly influenced by the affinity of the receptor for IP 3.