Dynamic clustering of IP3 receptors by IP3 Taufiq Rahman1 Pembroke College, Cambridge CB2 1RF, U.K., and Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, U.K.
Early Career Research Award Delivered at the Biochemical Society Centenary Event held at the Royal Society, London, on 16 December 2011 Taufiq Rahman
Abstract The versatility of Ca2 + as an intracellular messenger stems largely from the impressive, but complex, spatiotemporal organization of the Ca2 + signals. For example, the latter when initiated by IP3 (inositol 1,4,5-trisphosphate) in many cells manifest hierarchical recruitment of elementary Ca2 + release events (‘blips’ and then ‘puffs’) en route to global regenerative Ca2 + waves as the cellular IP3 concentration rises. The spacing of IP3 Rs (IP3 receptors) and their regulation by Ca2 + are key determinants of these spatially organized Ca2 + signals, but neither is adequately understood. IP3 Rs have been proposed to be pre-assembled into clusters, but their composition, geometry and whether clustering affects IP3 R behaviour are unknown. Using patch-clamp recording from the outer nuclear envelope of DT40 cells expressing rat IP3 R1 or IP3 R3, we have recently shown that low concentrations of IP3 cause IP3 Rs to aggregate rapidly and reversibly into small clusters of approximately four IP3 Rs. At resting cytosolic Ca2 + concentrations, clustered IP3 Rs open independently, but with lower open probability, shorter open duration and lesser IP3 sensitivity than lone IP3 Rs. This inhibitory influence of clustering on IP3 R is reversed when the [Ca2 + ]i (cytosolic free Ca2 + concentration) increases. The gating of clustered IP3 Rs exposed to increased [Ca2 + ]i is coupled: they are more likely to open and Key words: elementary calcium signal, inositol 1,4,5-trisphosphate, inositol 1,4,5-trisphosphate receptor (IP3 R), receptor clustering. Abbreviations used: [Ca2 + ]c , cytosolic Ca2 + concentration; [Ca2 + ]i , intracellular free Ca2 + concentration; CICR, Ca2 + -induced Ca2 + release; ER, endoplasmic reticulum; FKBP, FK506binding protein; IP3 , inositol 1,4,5-trisphosphate; [IP3 ], IP3 concentration; IP3 R, IP3 receptor; PLC, phospholipase C; RyR, ryanodine receptor; TIRFM, total internal reflection fluorescence microscopy. 1 email
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Biochem. Soc. Trans. (2012) 40, 325–330; doi:10.1042/BST20110772
close together, and their simultaneous openings are prolonged. Dynamic clustering of IP3 Rs by IP3 thus exposes them to local Ca2 + rises and increases their propensity for a CICR (Ca2 + induced Ca2 + rise), thereby facilitating hierarchical recruitment of the elementary events that underlie all IP3 -evoked Ca2 + signals.
Early Career Research Award
Early Career Research Award
Introduction Every cell needs to cope with its ever changing immediate environment marked by continuous arrival and disappearance of numerous stimuli. Although a rich palette of surface receptors are employed to recognize these exogenous signals of diverse nature, it is highly intriguing that only a handful of intracellular messengers are produced upon transduction of these signals. Perhaps even more impressive is how these messengers commit themselves to trigger (or modulate) appropriate cellular responses without compromising the fidelity. The latter is best epitomized by the universality and versatility of intracellular Ca2 + signals which regulate almost every aspect of cellular life, ranging from fertilization to cell death for every life form [1]. A substantial amount of cellular energy is continuously spent in keeping the basal [Ca2 + ]i (intracellular free Ca2 + concentration) at a low level (100–300 nM) and as such also maintaining a large concentration gradient of this ion towards cytosol with respect to internal Ca2 + stores and the extracellular compartment. Transient elevation of [Ca2 + ]i from the resting low level to a much higher (∼1 μM) level is what effectively constitutes a ‘Ca2 + signal’ [1]. Although such a rise in [Ca2 + ]i occurs on demand, the way it is triggered varies between cells largely depending on the presence or absence of excitable membranes. For the excitable cells such as neurons and those of skeletal muscle, Ca2 + signals are triggered by Ca2 + entry through some voltage-gated highly Ca2 + -selective ion channels and, to some extent, ligandgated Ca2 + -permeant channels expressed in their plasma membrane. Non-excitable cells (e.g. lymphocytes, acinar cells of exocrine glands, endothelial cells of blood vessels or fibroblasts) essentially lack this pathway and, following surface stimulation, they instead produce a few soluble second messengers such as IP3 (inositol 1,4,5-trisphosphate) C The
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that mobilize Ca2 + from intracellular stores such as the ER (endoplasmic reticulum). IICR (IP3 -evoked Ca2 + release) eventually depletes the ER and thus triggers further and more sustained Ca2 + entry via the store-operated pathway in the plasma membrane. I have been particularly interested in IP3 which is the most widely used Ca2 + -mobilizing intracellular messenger and, in the rest of the present article, I focus on IP3 -evoked Ca2 + signalling.
IP3 -evoked Ca2 + signals are spatially organized IP3 is produced at the plasma membrane as a result of hydrolysis of PIP2 (phosphatidylinositol 4,5-bisphosphate) in a PLC (phospholipase C)-dependent manner. Activation of PLC occurs when cell-surface receptors (receptor tyrosine kinases and those coupled to the Gq/11 family of G-proteins) are stimulated by various hormones, growth factors or neurotransmitters. Upon production, IP3 rapidly diffuses into the cytosol and binds to IP3 Rs (IP3 receptors) that are mainly expressed in the membranes of the ER, the major internal Ca2 + store. IP3 binding to IP3 Rs causes the pore to open, through which Ca2 + rapidly flows into the cytosol down its concentration gradient. Cells often have numerous and diverse types of surface receptors, many of which, upon stimulation, activate PLC and thus converge on making the same intracellular messenger, IP3 , and the Ca2 + signalling that ensues. Yet diverse, but specific, cellular effects are elicited without any chaos. Indeed, Ca2 + signals triggered by IP3 have been specifically implicated in fertilization, proliferation and differentiation, metabolism, contraction, fluid secretion, exocytosis, chemotaxis, platelet aggregation, synaptic plasticity and opening of some Ca2 + -activated ion channels [2]. So what imparts such enormous versatility in the action of Ca2+ ? Growing evidence suggests that this versatility stems largely from the spatiotemporal variations in Ca2 + signals as well as the availability of proteins that can ‘interpret’ different messages encoded in different spatiotemporal patterns of Ca2 + signals. IP3 -evoked Ca2 + signals in most intact cells do manifest complex spatiotemporal patterns such as waves and oscillations that result from the hierarchical recruitment of elementary Ca2 + -release events [3–5]. Stimulation of the cell-surface receptors with graded concentrations of IP3 -generating stimuli triggers Ca2 + signals of differing amplitudes and spatial dimensions, sequentially growing from elementary to global Ca2 + release events [6] (Figure 1). When the intracellular [IP3 ] (IP3 concentration) is low, the fundamental events known as ‘Ca2 + blips’ occur sporadically. These are small localized elevations of [Ca2 + ]i and are most likely to stem from random openings of single IP3 Rs. Such events typically last less than 130 ms, and have amplitudes of less than 40 nM. As [IP3 ] increases with higher stimulus intensity, more IP3 Rs become active, and Ca2 + mobilized through a group of active IP3 Rs leads to the appearance of ‘elementary or intermediate events’ known as ‘Ca2 + puffs’. The latter remain localized, spreading no more than 6 μm, C The
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Figure 1 Hierarchical recruitment of IP3 -mediated Ca2 + signalling events (A) At low [IP3 ], the fundamental event, also known as a ‘Ca2 + blip’ occurs, reflecting the Ca2 + signal produced by a single active IP3 R. (B) As the [IP3 ] increases, more IP3 Rs within a cluster bind IP3 , and Ca2 + released from the first activated channel activates its neighbours through a CICR mechanism. A ‘Ca2 + puff’ is produced in this way. (C) With even further increase in the [IP3 ], more IP3 Rs bind IP3 , more ‘puffs’ are produced and spatiotemporal summation of all puffs eventually results in a global Ca2 + wave. This Figure is based on existing notion that IP3 Rs exist as pre-formed clusters within the ER membrane.
have amplitudes of 50–600 nM and last for ∼1 s. With much higher [IP3 ], more and more puffs are ignited, and the spatiotemporal summation of all puffs eventually leads to a global regenerative Ca2 + wave that spreads throughout the cell [7–9]. It is important to appreciate the fact that the elementary Ca2 + -release events are not just generic building blocks for global Ca2 + waves. These spatially restricted Ca2 + signals can be delivered locally to some target proteins and thus can regulate distinct cellular processes [3]. It is therefore imperative to have a better understanding of what makes spatial organization of Ca2 + signals possible. For IP3 -evoked Ca2 + signals, the sequential progression of signal Ca2 + from an elementary to a global level seems to capitalize on at least two critical processes that are directly related to IP3 Rs. First, IP3 tunes the Ca2 + -sensitivity of IP3 Rs in such a way that Ca2 + released from an active IP3 R can activate another IP3 R through a process known as CICR (Ca2 + -induced Ca2 + release) [10,11]. In order
Metabolic sensors and their interplay with cell signalling and transcription
for CICR to operate successfully, it is intuitively obvious that the process should also be dependent on, apart from the cytoplasmic diffusibility of Ca2 + itself governed by the nature of existing buffers, the average distance Ca2 + needs to traverse before it encounters the next IP3 R. The latter is dictated by the distribution and spatial arrangement of IP3 Rs within the ER membrane. Review of the existing literature on IP3 Rs is suggestive of non-random distribution of these channels within the native ER membrane of various cell types [12]. Detection of Ca2 + puffs using single-cell Ca2 + imaging itself implies the existence of IP3 R clusters [8,9,13]. Confocal microscopy in several cell types confirmed that both native and tagged IP3 Rs form clusters [14–18]. Patch-clamp recordings have also indicated heterogeneous distribution of IP3 Rs in the outer nuclear membrane that is continuous with the ER [19–21]. The prevailing view is thus in favour of IP3 Rs existing as pre-formed static clusters in the ER membrane and within each cluster, IP3 Rs are stimulated by CICR initiated by a Ca2 + blip. Our results have led us to suggest that IP3 Rs may assemble ‘on demand’ into clusters in response to IP3 and the process is dynamically regulated [22,23].
What prompts IP3 Rs to cluster? The first clue regarding the possible mechanism of IP3 R clustering came from Wojcikiewicz and colleagues [17], showing that agonists triggered redistribution of IP3 Rs (type 2 and 3 predominantly) in some cells. This could be mimicked by increasing [Ca2 + ]c (cytosolic Ca2 + concentration), using either ionomycin or thapsigargin, and it could be prevented by removal of extracellular Ca2 + . Thus an increase in [Ca2 + ]c was suggested to be the trigger for clustering [17]. However, in a later study, the Mikoshiba group [15] observed that agonist-evoked clustering of tagged IP3 Rs coincided with a peak in IP3 production, with no obvious correlation with the cytosolic Ca2 + transients, and a PLC inhibitor (U73122) could suppress the ionophore-induced IP3 R clustering observed by Wilson et al. [17]. Furthermore, mutant IP3 Rs unable to change conformation upon IP3 binding did not cluster [22]. All of these led to the suggestion that the critical trigger for IP3 R clustering was IP3 binding to IP3 Rs followed by the necessity of the subsequent conformational transition of these channels to their open state [15,18]. We found that IP3 Rs recorded from the outer nuclear membranes of DT40 cells expressing recombinant IP3 Rs (isoform 1 and 3) are initially randomly distributed, but a low [IP3 ] (lower than required to maximally activate them,
