Linking structure to function: Recent lessons from inositol 1,4,5-trisphosphate receptor mutagenesis

Abstract

Great insight has been gained into the structure and function of the inositol 1,4,5 trisphosphate receptor (InsP(3)R) by studies employing mutagenesis of the cDNA encoding the receptor. Notably, early studies using this approach defined the key constituents required for InsP(3) binding in the N-terminus and the membrane spanning regions in the C-terminal domain responsible for channel formation, targeting and function. In this article we evaluate recent studies which have used a similar approach to investigate key residues underlying the in vivo modulation by select regulatory factors. In addition, we review studies defining the structural requirements in the channel domain which comprise the conduction pathway and are suggested to be involved in the gating of the channel.

Figure 1. Consensus PKA phosphorylation substrate motifs and “Walker type A” ATP binding motifs in the InsP₃R isoforms

The general domain structure of InsP₃R family members is shown for S2⁺ InsP₃R1 in A; S2^- InsP₃R-1 in B; InsP₃R-2 in C and InsP₃R-3 in D. In the schematic for each receptor PKA phosphorylation sites are shown above and putative ATP binding domains are shown below. Residues colored red indicate amino acids which when mutated have functional consequences. Residues colored in green indicate amino acids which when mutated did not result in effects on InsP₃R activity. Residues within the putative ATP binding domains underlined in red indicate residues which abrogate binding of ATP to GST-fusion proteins containing the particular site. In InsP₃R-1 (A and B) the PKA phosphorylation sites Serine 1589 and Serine 1755 are separated by the S2 splice region shown in blue (amino acids 1692-1732) in the top panel. In B the blue underlined residues indicate the N and C terminal boundaries of the S2 splice region. The site of the in frame deletion present in the Opthisthotonos mouse (OPT; amino acids 1732-1839) which results in deletion of a phosphorylation site and the ATPA site is indicated. Below the schematic is a diagram of the fragments of InsP₃R-1 produced by limited digestion with trypsin [46, 126, 127]. ATP binding was reported in the C-terminal 40 kDa and 91 kDa fragments [46]. In B, PKA sites and the complement of ATP binding motifs are shown in the S2^- InsP₃R-1. The amino acid numbering is based on the rat S2⁺ InsP₃R-1. In C, a functionally important PKA phosphorylation site at Serine 937 is shown. The ATPB Walker motif is also functionally relevant in InsP₃R-2. Below the schematic a diagram is shown which illustrates a subcloning strategy used to determine phosphorylation sites in InsP₃R-2. The fragments of InsP₃R2 were based on tryptic sites in InsP₃R-1. PKA phosphorylation of the 76 kDa fragment was demonstrated [45]. In D, phosphorylation sites Serine 916, Serine 934 and Serine 1832 are shown, together with the ATPB Walker motif. Mutation of each of these sites appears to be without functional consequence. Below the schematic is a diagram depicting fragments produced by limited chymotryptic digestion of InsP₃R-3 [43, 46]. ATP binding was demonstrated in the C-terminal 95 kDa fragment [46]. PKA dependent phosphorylation was shown in the N terminal 105 kDa fragment; the 70 kDa and 35 kDa fragments [43].

Figure 2. The pore and C-terminal tail of InsP₃ receptors

Panel A shows the sequence of a portion of the pore region of InsP₃Rs. The consensus residues shown in bold are identical in all 3 InsP₃R isoforms and all 3 RyR isoforms although only the sequence of RyR1 is shown in the Figure. Residues which have been mutated in InsP₃R1 are shown together with their functional consequences. The analogous mutations in RyRs are shown. For additional information see text and Ref. (9). The R2596 position is indicated by an asterisk. Panel B A homology model of the InsP₃R based on a KirBac 1.1 template is shown. The image is taken from Ref. (9) and is used with permission. Only the location of selected mutations from Panel A are shown. The linkers between S5 and the pore helix, and between the selectivity filter and S6, were not included in the model. Panel C shows a cartoon of the C-terminal tail with endogenous cysteines indicated by open circles and bars to indicate the approximate location of interacting proteins. CC = coiled coil domain. GIT; G-protein coupled receptor kinase-interacting protein, PP1-a; protein phosphates 1-a, 4.1N; neuron specific isoform of erythrocyte protein band 4.1. For additional details see text.