Functional Determination of Calcium Binding Sites Required for the Activation of Inositol 1,4,5-trisphosphate receptors

Abstract

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca2+) signals in response to various external cues. Notably, IP3R channel activity is determined by several obligatory factors including IP3, Ca2+ and ATP. The critical basic amino acid residues in the N-terminal IP3-binding core (IBC) region that facilitate IP3 binding are well characterized. In contrast, the residues conferring the biphasic regulation by Ca2+ are yet to be ascertained. Using comparative structural analysis of Ca2+ binding sites identified in two main families of intracellular Ca2+-release channels, ryanodine receptors (RyRs) and IP3Rs, we identified putative acidic residues coordinating Ca2+ in the cytosolic calcium sensor region in IP3Rs. We determined the consequences of substituting putative Ca2+ binding, acidic residues in IP3R family members. We show that the agonist-induced Ca2+ release, single channel open probability (P0) and Ca2+ sensitivities are markedly altered when the negative charge on the conserved acidic side chain residues are neutralized. Remarkably, neutralizing the negatively charged side chain on two of the residues individually in the putative Ca2+ binding pocket shifted the Ca2+ required to activate IP3R to higher concentrations, indicating that these residues likely are a component of the Ca2+ activation site in IP3R. Taken together, our findings indicate that Ca2+ binding to a well conserved activation site is a common underlying mechanism resulted in increased channel activity shared by IP3Rs and RyRs.

Structural dynamics underlying gating and regulation in IP3R channel

“…Moreover, alignment of 3D structures of the Ca-III S site in IP 3 R1, IP 3 R3 and RyR channel confirms a structural conservation of this Ca 2+ binding site across both families of Ca 2+ release channels as demonstrated in our previous studies 30,55 . In our companion study, mutations of these residues markedly affected single channel activity, such that altering the negative charge on either aspartate residue, shifted the [Ca 2+ ] dependence for activation to the left, consistent with an important role of this site in Ca 2+ activation of IP 3 R1 activity 30 . A conserved E2101 (E4033 in RyR1 and E2005 in IP 3 R3) was previously proposed to be responsible for Ca 2+ sensitivity for activation of IP 3 R1 and RyR1 channels 56,57 , however, this residue does not directly contribute to coordination of a Ca 2+ ion in the binding pocket, and appears to exert an effect via allosteric interactions with surrounding residues.…”

“…Noteworthy, none of the cytosolic Ca 2+ binding sites described here is similar to that of helix-turn-helix (EF-hand) Ca 2+ -binding proteins. Moreover, alignment of 3D structures of the Ca-III S site in IP 3 R1, IP 3 R3 and RyR channel confirms a structural conservation of this Ca 2+ binding site across both families of Ca 2+ release channels as demonstrated in our previous studies 30,52 . In our companion study, mutations of these residues markedly affected single channel activity, such that altering the negative charge on either aspartate residue, shifted the [Ca 2+ ] dependence for activation to the left, consistent with an important role of this site in Ca 2+ activation of IP 3 R1 activity 30 .…”

“…In addition, we used a deep neural network approach 28 and 3D variability analysis 29 to extract dynamic features of the IP3R1 structure directly from 2D cryo-EM images. Our structural findings and hypothesis were validated through mutagenesis and electrophysiology (see also 30 ). From these studies, we correlate protein conformational changes that connect the ligand-binding regulatory sites to the channel gate.…”

“…Moreover, our recent studies demonstrate that mutations of residues in the Ca-III S site alter channel activity. Specifically, neutralizing the negatively charged side chains of either E1978 or E2042 in the Ca-III S site shifted the Ca 2+ to higher concentrations required to activate IP 3 R1 30 . This indicates that these residues are likely components of the Ca 2+ activation site in IP 3 R1.…”

“…Consistent with these data, mutagenesis of amino acid residues in the Ca-III S site alters channel activity. Specifically, neutralizing the negatively charged side chain on two of the residues individually in Ca-III S shifted the Ca 2+ required to activate IP 3 R to higher concentrations, indicating that these residues are likely components of the Ca 2+ activation site in IP 3 R1 30 . As it might have been anticipated, binding of IP 3 "tunes" Ca 2+ -inhibition of the IP 3 R channel 16,31,32,[51][52][53] .…”