Calcium regulation of inositol 1,4,5-trisphosphate receptors.
Ca2+ exerts both a stimulatory and inhibitory effect on type-I IP3R channel activity. However, the structural determinants of Ca2+ sensing in IP3Rs are not fully understood. Previous studies by others have identified eight domains of the type-I IP3R that bind 45Ca2+ 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 IP3R and measured the Ca2+ regulation of IP3-mediated Ca2+ release in COS-7 cells. 45Ca2+ flux assays measured with a maximal [IP3] (1 microM) indicate that one of the mutants retained a Ca2+ sensitivity that was not significantly different from control (E411Q), three of the mutants show an enhanced Ca2+ inhibition (D426N, E428Q and E439Q) and two of the mutants were relatively insensitive to Ca2+ inhibition (D442N and D444N). IP3 dose-response relationships indicated that the sensitivity to Ca2+ inhibition and affinity for IP3 were correlated for three of the constructs. Other mutants with enhanced IP3 sensitivity (e.g. R441Q and a type-II/I IP3R chimera) were also less sensitive to Ca2+ inhibition. We conclude that the acidic residues within the aa378-450 segment are unlikely to represent a single functional Ca2+ binding domain and do not contribute to Ca2+ 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 IP3R isoforms may display a range of Ca2+ sensitivities that are determined by multiple sites within the protein and markedly influenced by the affinity of the receptor for IP3.
Joseph
Suresh K
SK
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Rm. 230A JAH, 1020 Locust Street, Philadelphia, PA 19107, USA. Suresh.Joseph@mail.tju.edu
Brownell
Samuel
S
Khan
M Tariq
MT
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D024342:Amino Acids, Acidic / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism
D024342:Amino Acids, Acidic; D002135:Calcium-Binding Proteins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D020033:Protein Isoforms; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium