Jianwei Shuai

Ca 2+ Puffs Originate from Preestablished Stable Clusters of Inositol Trisphosphate Receptors

Localized calcium signals called Ca2+ puffs arise at preestablished clusters of IP3Rs. No Assembly Required The second messenger inositol trisphosphate (IP3) acts on IP3 receptors (IP3Rs) in the endoplasmic reticulum (ER) membrane to elicit Ca2+ release from intracellular stores, enabling the generation of intracellular Ca2+ signals from extracellular messengers. The regulation of these Ca2+ signals depends on the clustered organization of IP3R. Using optical techniques capable of recording single-channel IP3R activity in intact mammalian cells, Smith et al. explored the dynamics of IP3-dependent Ca2+ signals and concluded that local Ca2+ signals (known as puffs) arise from preassembled clusters of IP3R rather than depending on rapid and dynamic clustering in response to the IP3 signal itself. Intracellular calcium ion (Ca2+) signaling crucially depends on the clustered organization of inositol trisphosphate receptors (IP3Rs) in the endoplasmic reticulum (ER) membrane. These ligand-gated ion channels liberate Ca2+ to generate local signals known as Ca2+ puffs. We tested the hypothesis that IP3 itself elicits rapid clustering of IP3Rs by using flash photolysis of caged IP3 in conjunction with high-resolution Ca2+ imaging to monitor the activity and localization of individual IP3Rs within intact mammalian cells. Our results indicate that Ca2+ puffs arising with latencies as short as 100 to 200 ms after photorelease of IP3 already involve at least four IP3R channels, and that this number does not subsequently grow. Moreover, single active IP3Rs show limited mobility, and stochastic simulations suggest that aggregation of IP3Rs at puff sites by a diffusional trapping mechanism would require many seconds. We thus conclude that puff sites represent preestablished, stable clusters of IP3Rs and that functional IP3Rs are not readily diffusible within the ER membrane.

Modeling Ca2+ Feedback on a Single Inositol 1,4,5-Trisphosphate Receptor and Its Modulation by Ca2+ Buffers

The inositol 1,4,5-trisphosphate receptor/channel (IP(3)R) is a major regulator of intracellular Ca(2+) signaling, and liberates Ca(2+) ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP(3) and Ca(2+). Although the steady-state gating properties of the IP(3)R have been extensively studied and modeled under conditions of fixed [IP(3)] and [Ca(2+)], little is known about how Ca(2+) flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca(2+) binding sites. We thus simulated the dynamics of Ca(2+) self-feedback on monomeric and tetrameric IP(3)R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca(2+) buffers that slow the collapse of the local [Ca(2+)] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca(2+) to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca(2+) binding site on the IP(3)R in relation to the channel pore.

Calcium Domains around Single and Clustered IP3 Receptors and Their Modulation by Buffers

We study Ca(2+) release through single and clustered IP(3) receptor channels on the ER membrane under presence of buffer proteins. Our computational scheme couples reaction-diffusion equations and a Markovian channel model and allows our investigating the effects of buffer proteins on local calcium concentrations and channel gating. We find transient and stationary elevations of calcium concentrations around active channels and show how they determine release amplitude. Transient calcium domains occur after closing of isolated channels and constitute an important part of the channels feedback. They cause repeated openings (bursts) and mediate increased release due to Ca(2+) buffering by immobile proteins. Stationary domains occur during prolonged activity of clustered channels, where the spatial proximity of IP(3)Rs produces a distinct [Ca(2+)] scale (0.5-10 microM), which is smaller than channel pore concentrations (>100 microM) but larger than transient levels. While immobile buffer affects transient levels only, mobile buffers in general reduce both transient and stationary domains, giving rise to Ca(2+) evacuation and biphasic modulation of release amplitude. Our findings explain recent experiments in oocytes and provide a general framework for the understanding of calcium signals.

An investigation of models of the IP3R channel in Xenopus oocyte.

We consider different models of inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) channels in order to fit nuclear membrane patch clamp data of the stationary open probability, mean open time, and mean close time of channels in the Xenopus oocyte. Our results indicate that rather than to treat the tetrameric IP(3)R as four independent and identical subunits, one should assume sequential binding-unbinding processes of Ca(2+) ions and IP(3) messengers. Our simulations also favor the assumption that a channel opens through a conformational transition from a close state to an active state.

The dynamics of small excitable ion channel clusters

Through computational modeling we predict that small sodium ion channel clusters on small patches of membrane can encode electric signals most efficiently at certain magic cluster sizes. We show that this effect can be traced back to algebraic features of small integers and are universal for channels with a simple gating dynamics. We further explore physiologic conditions under which such effects can occur.