OP
TreasureVibe
Member
- Joined
- Jul 3, 2016
- Messages
- 1,941
IP3 Receptors: Toward Understanding Their ActivationSo there exists a specific protein, indisputably a membrane pore large enough to admit calcium yet not specifically; this will allow passage of both larger and smaller ions:
'The IP₃ receptor, when activated, can conduct all four alkaline earth cations with conductances in the order of Ba²⁺ > Sr²⁺ > Ca²⁺ > Mg²⁺.' ―Yoshida
While true that this particular membrane pore binds inositol triphosphate (KD ≈ 2–100·nM) it also binds ATP, calcineurin, FKBP12, and some variants will bind calmodulin.
'As shown in Fig. 1, the domain contains putative binding sites for various modulators of the channel such as ATP, Ca²⁺, calmodulin, FK506 binding protein 12 (FKBP 12)...' ―Yoshida
'A specific binding site for ATP was detected in purified type 1 IP₃ receptor subunit (65), and two consensus sequences for ATP binding site were found in its amino acid sequence (24, 25), both located in the coupling domain of the receptor.' ―Yoshida
Yutaka Yoshida isn't a physicist, and doesn't even attempt to describe how calcium actually enters the cell; review articles never prove anything, and its common for their authors to be overly-equitable and to not express strong beliefs (lest it turn into a theoretical article). Yet he does cite another review article in his section marked 'IV. Function of IP₃ receptor.'
Bezprozvanny, I. "The inositol 1,4,5-trisphosphate (InsP₃) receptor." The Journal of membrane biology (1995)
'The ability of Mg²⁺ ions to carry substantial currents through these channels is especially striking when the very high hydration energy and extremely slow substitution rate of water molecules in the inner hydration shell of Mg²⁺ ions (Hille, 1992) is taken into consideration. One possible explanation of this observation is that when Mg²⁺ ions pass through the selectivity filters of both intracellular Ca²⁺ channels they are able to keep the inner shell of water molecules. This suggestion implies that the narrowest portion of the channel pore should be at least 10·Å for both channels. An even larger estimate of the pore size (40·Å) was obtained for the RyR (Lindsay et al., 1991) based on the ability of large organic cations like Tris⁺ and TEA⁺ to permeate through these channels.' ―BezprozvannyThis is a nonspecific channel, and the author seems to doubt the existence of selective calcium channels:
'It could be concluded from the studies of InsP₃R (Bezprozvanny & Ehrlich, 1994) and RyR (Lindsay et al., 1991; Tinker & Williams, 1992) permeation that both channels are rather nonspecific cation selective channels, permeable to Ca²⁺ and monovalent cations.' ―Bezprozvanny
'As an aside, it follows from this discussion that if plasma membrane InsP₃-gated Ca²⁺-selective channels do exist (Kuno & Gardner, 1987; Fujimoto et al., 1992) they must be much more selective for divalent cations than intracellular InsP₃R.' ―BezprozvannyThe author notes the affinity ATP has for this membrane pore, and also confirms the ionic series of permissivity:
'These authors came to the conclusion that ATP was a necessary cofactor for the activation of what was then a hypothetical InsP₃R (Smith et al., 1985). The role of ATP as an allosteric activator of the InsP₃R was proposed later based on experiments with receptor that was purified and reconstituted into liposomes (Ferris et al., 1990). It was found that 10 gM ATP or nonhydrolyzable ATP analogues dramatically potentiated InsP₃-mediated Ca²⁺ flux into vesicles containing purified InsP₃R. The existence of a specific ATP-binding site on the InsP₃R was also demonstrated in the same report (Ferris et al., 1990).' ―Bezprozvanny
'All four alkaline earth cations tested were able to pass through the InsP₃R with single channel conductances that fall in the sequence Ba > Sr > Ca > Mg. The same order of conductances was reported for the RyR (Tinker & Williams, 1992) although the absolute values of the single channel conductance are approximately twice as large for the RyR.' ―BezprozvannyBased on these considerations the inositol triphosphate receptor could just as easily be called the 'ATP barium pore,' the 'membrane FKBP12 receptor,' or the 'membrane nonselective ion channel.' Adenosine triphosphate (ATP) is known for it's propensity for complexing magnesium, which can also pass through the pore, yet will chelate calcium in its absence. The physical forces responsible for determining how much Ca²⁺ enters and exits through this pore had gone unexplained by both of these authors, and no mention of the fact that inositol phosphates chelate calcium. The closest thing to a physical explanation had been the mention of the luminal 'glutamate ring,' an amino acid often post-translationally modified to γ-carboxyglutamate—a calcium chelator and how vitamin K ultimately exerts its calcemic functions.
I would guess that this is simple a pore having no directionality, allowing ions either in or out just the same; it's complete nonspecificity towards both the 'activating' ligand and the ions permitted is freely admitted, and even a simple dialysis bag will allow unidirectional ion flow if a chelator is placed on one side (i.e. EDTA, ATP, IP₃). An experimental study I would like to see conducted would a comparison of Ca²⁺ flux after the addition of inositol triphosphate, adenosine triphosphate, ethylene diamine tetra-acetate, and simple pyrophosphate. I would think any membrane study that makes claims about the ability of a protein to actually cause unidirectional ion flow would need something akin to an 'EDTA dialysis model' serving as a control: Only then can you eliminate underlying dialysis forces from the equation, if they are not significant, or subtract them from the 'receptor-driven flux' if the are; that could prove its ontological status, but so far I have seen nothing to indicate that its anything more than merely an ion pore.
Colin W. Taylor and Stephen C. Tovey
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, United Kingdom
Correspondence:Email: ku.ca.mac@0001twc
2010
Abstract
Inositol 1,4,5-trisphosphate receptors (IP3R) and their relatives, ryanodine receptors, are the channels that most often mediate Ca2+ release from intracellular stores. Their regulation by Ca2+ allows them also to propagate cytosolic Ca2+ signals regeneratively. This brief review addresses the structural basis of IP3R activation by IP3 and Ca2+. IP3 initiates IP3R activation by promoting Ca2+ binding to a stimulatory Ca2+-binding site, the identity of which is unresolved. We suggest that interactions of critical phosphate groups in IP3 with opposite sides of the clam-like IP3-binding core cause it to close and propagate a conformational change toward the pore via the adjacent N-terminal suppressor domain. The pore, assembled from the last pair of transmembrane domains and the intervening pore loop from each of the four IP3R subunits, forms a structure in which a luminal selectivity filter and a gate at the cytosolic end of the pore control cation fluxes through the IP3R.
A BRIEF HISTORY OF IP3 RECEPTORS
Sidney Ringer, in his famous correction to an earlier paper, showed that Ca2+ entry can evoke a physiological response by demonstrating that beating of the frog heart requires extracellular Ca2+ (Ringer 1883). Almost a century passed before it became clear that this Ca2+ entry, via voltage-gated Ca2+channels, was not directly responsible for contraction, but instead provided the trigger for a much larger release of Ca2+ from stores within the sarcoplasmic reticulum (SR). The latter is mediated by type-2 ryanodine receptors (RyR) (Fabiato 1983; Cheng et al. 1993), which like many Ca2+ channels, are able both to transport Ca2+ through an open pore and respond to it. These observations highlight two general points. First, cells call upon two sources of Ca2+ to evoke increases in cytosolic Ca2+ concentration; second, interactions between these Ca2+ fluxes across the plasma membrane and the membranes of intracellular stores are important determinants of the physiological response. The same points apply to the Ca2+ signals evoked by receptors that stimulate phospholipase C (PLC) and, thereby, formation of inositol 1,4,5-trisphosphate (IP3).
The biochemical sequence linking these receptors to formation of IP3 emerged in the 1980s (Michell et al. 1989; Berridge 2005), but work in the decade before had established that many receptors regulate many different responses by increasing the cytosolic Ca2+ concentration (Rasmussen 1970; Berridge 1975). In his influential review, Bob Michell (Michell 1975), building on work showing that many of these receptors also stimulate phospholipid turnover (Hokin and Hokin 1953), had suggested a causal link between phosphoinositide hydrolysis and Ca2+ signals. Here, as in many studies, the emphasis was on Ca2+ entry, with a consensus only slowly emerging that Ca2+ fluxes across both the plasma membrane and the membranes of intracellular stores contribute to cytosolic Ca2+ signals (Rasmussen 1970; Berridge 1975; Williams 1980; Putney et al. 1981). In the years following Michell’s review, decisive evidence, much of it coming from Mike Berridge’s elegant studies of blowfly salivary gland, established that phosphoinositide hydrolysis is, as predicted by Michell, required for PLC-linked receptors to evoke Ca2+ signals (Berridge and Fain 1979). The same preparation was used to show that IP3 is the first water-soluble product of the signaling pathway (Berridge 1983). IP3, thus, emerged as a prime candidate for the cytosolic messenger linking events at the plasma membrane to release of Ca2+ from intracellular stores. Paradoxically, it was to be many years before the links between receptors that stimulate PLC and Ca2+ entry were resolved. These came with elaboration of the pathways linking empty Ca2+ stores to Ca2+ entry, the so-called store-operated Ca2+ entry pathway (Putney 1997; Park et al. 2009), and recognition that many trp channels are regulated by products of PLC activity (Nilius et al. 2007). IP3 receptors (IP3R) also contribute more directly to Ca2+ entry across the plasma membrane either because, at least in some cells, IP3R are functionally expressed in the plasma membrane (Dellis et al. 2006; Dellis et al. 2008), or perhaps through their direct interactions with other plasma membrane Ca2+ channels (Kiselyov et al. 1999). Here, we focus solely on Ca2+ release from the endoplasmic reticulum (ER) by IP3R. Some of the key steps in the evolution of our current understanding of IP3R are listed in Table 1.
IP3 Receptors: Toward Understanding Their Activation