Diffusion-trap Mechanism Research Articles

Store-Operated Calcium Channels: From Function to Structure and Back Again.

Store-operated calcium (Ca2+) entry (SOCE) occurs through a widely distributed family of ion channels activated by the loss of Ca2+ from the endoplasmic reticulum (ER). The best understood of these is the Ca2+ release-activated Ca2+ (CRAC) channel, which is notable for its unique activation mechanism as well as its many essential physiological functions and the diverse pathologies that result from dysregulation. In response to ER Ca2+ depletion, CRAC channels are formed through a diffusion trap mechanism at ER-plasma membrane (PM) junctions, where the ER Ca2+-sensing stromal interaction molecule (STIM) proteins bind and activate hexamers of Orai pore-forming proteins to trigger Ca2+ entry. Cell biological studies are clarifying the architecture of ER-PM junctions, their roles in Ca2+ and lipid transport, and functional interactions with cytoskeletal proteins. Molecular structures of STIM and Orai have inspired a multitude of mutagenesis and electrophysiological studies that reveal potential mechanisms for how STIM is toggled between inactive and active states, how it binds and activates Orai, and the importance of STIM-binding stoichiometry for opening the channel and establishing its signature characteristics of extremely high Ca2+ selectivity and low Ca2+ conductance.

My research life: from synaptic transmission to behavior

I have studied signal transmission at synapses and the effects of drugs on it at the molecular and cellular levels. Specific areas of research interest are outlined here. 1) Electrophysiological experiments in cats and rabbits suggested that a new type of analgesic, the phenothiazine derivative levomepromazine, exerts analgesic effects by depressing emotional responses accompanying the sensation of pain. 2) It was hypothesized that motoneurons had long-term effects on muscle cell membrane properties, in addition to controlling moment-to-moment activities. The substance to recover the post-denervation changes in muscle properties in culture was partially purified from mouse nerve extract, which suggested that trophic influences were exerted by substances released from motoneurons. 3) Muscles innervated by adrenergic fibers had sites responsive to acetylcholine as well as to adrenaline in early life in chicks, but only the adrenaline-responsive sites remained during development. Acetylcholine receptor clusters on Xenopus muscles were concentrated at the cholinergic neuromuscular junctions by the movement of receptors from outside the junctions during development. The passive diffusion-trap mechanism explained the accumulation of synaptic receptors at synapses. 4) We found two endocytic pathways and pools of synaptic vesicles contributing to low- and high-frequency synaptic transmission at Drosophila nerve terminals. We then identified two Ca2+ channels designated for the low- and high-frequency endocytosis of synaptic vesicles, straightjacket Ca2+ channels in the active zone and La3+-sensitive Ca2+ channels in the inactive zone at the terminals, respectively. Recently, Drosophila melanogaster has been used as a model for studying the social brain, and the heat avoidance response of the flies was found to be socially enhanced. Future studies are expected to reveal mechanisms underlying social brain functions at the gene level.

Stoichiometric requirements for trapping and gating of Ca 2+ release-activated Ca 2+ (CRAC) channels by stromal interaction molecule 1 (STIM1)

Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of ∼2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.

Stoichiometric Requirements for Trapping and Activation of CRAC Channels by STIM1 At ER-Plasma Membrane Junctions

Store-operated Ca2+ entry is regulated by physical interactions of the ER Ca2+ sensor STIM1 and the CRAC channel protein Orai1. Recent studies support a diffusion-trap mechanism in which Ca2+ depletion from the ER causes STIM1 to accumulate at ER-plasma membrane (ER-PM) junctions, where its CRAC activation domain (CAD) binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. The ability of the CAD to cluster CRAC channels suggests that each channel contains multiple STIM1 binding sites, but the number of sites that must be occupied to trap and activate CRAC channels is unknown. By competing for a limited amount of STIM1, increasing levels of Orai1 are expected to reduce the number of STIM1s bound per channel until the minimum stoichiometry sufficient for channel trapping is reached. In HEK293 cells expressing a fixed amount of mCherry-STIM1 and increasing levels of GFP-Orai1, the junctional STIM1:Orai1 ratio reached a minimum of ∼0.3; thus, binding of 1-2 STIM1 appears sufficient to immobilize tetrameric CRAC channels at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current (ICRAC) was a highly nonlinear bell-shaped function of Orai1 expression, and the minimum STIM1:Orai1 stoichiometry for channel trapping failed to evoke significant activation. A simple cooperative gating model fitted to the data suggests that while STIM1 binding to a single site is sufficient to trap CRAC channels, four sites must be occupied to cause significant activation. This highly nonlinear behavior supports conclusions based on current fluctuation analysis (Prakriya and Lewis, J. Gen. Physiol. 128:373-86, 2006) that the slow development of whole-cell ICRAC after store depletion reflects the stepwise recruitment of individual channels from a silent to a high open-probability state as they enter ER-PM junctional sites.

Allosteric changes of the NMDA receptor trap diffusible dopamine 1 receptors in spines.

The dopaminergic and glutamatergic systems interact to initiate and organize normal behavior, a communication that may be perturbed in many neuropsychiatric diseases, including schizophrenia. We show here that NMDA, by allosterically modifying NMDA receptors, can act as a scaffold to recruit laterally diffusing dopamine D1 receptors (D1R) to neuronal spines. Using organotypic culture from rat striatum transfected with D1R fused to a fluorescent protein, we show that the majority of dendritic D1R are in lateral diffusion and that their mobility is confined by interaction with NMDA receptors. Exposure to NMDA reduces the diffusion coefficient for D1R and causes an increase in the number of D1R-positive spines. Unexpectedly, the action of NMDA in potentiating D1R recruitment was independent of calcium flow via the NMDA receptor channel. Thus, a highly energy-efficient, diffusion-trap mechanism can account for intraneuronal interaction between the glutamatergic and dopaminergic systems and for regulation of the number of D1R-positive spines. This diffusion trap system represents a molecular mechanism for brain plasticity and offers a promising target for development of antipsychotic therapy.

Local accumulation of acetylcholine receptors is neither necessary nor sufficient to induce cluster formation

Acetylcholine receptors (AChRs) accumulate at developing neuromuscular junctions in part via lateral migration of diffusely expressed receptors. Using a model system--cultured Xenopus muscle cells exposed to electric fields--we have shown that AChRs, concentrated at the cathode-facing cell pole, continue to aggregate there after the field is terminated (Stollberg and Fraser, 1988). These observations are consistent with the possibility that the field-induced increase in receptor concentration triggers the aggregation event. Only 2 other molecular events could initiate the electric field-induced receptor aggregation: (1) a local increase in the density of some other molecules, or (2) a voltage-sensitive mechanism. Treatment of muscle cell cultures with neuraminidase changes the cell surface charge and has been reported to reverse the direction of electromigration for AChRs and concanavalin A binding sites (Orida and Poo, 1978). Using digitally analyzed fluorescence videomicroscopy, we find that AChRs in neuraminidase-treated cultures accumulate at both cell poles in an electric field. After termination of the field, the AChR continues to aggregate at the cathode-facing pole, as in cells not treated with neuraminidase. However, receptor density decreases at the anode-facing pole, indicating that elevated AChR density does not initiate receptor aggregation. Cells pretreated with neuraminidase and trypsin (which blocks receptor aggregation) display reversed receptor distributions compared to untreated controls, indicating that electromigration has indeed been reversed. The rate at which neuraminidase- and trypsin-treated cells approach steady-state distributions indicates a receptor diffusion constant of approximately 1.2 x 10(-9) cm2/sec, consistent with a diffusion trap mechanism of receptor aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Electric Field-Induced Redistribution of ACh Receptors on Cultured Muscle Cells: Electromigration, Diffusion, and Aggregation.

Xenopus myoball cultures were used to examine the lateral migration and clustering of acetylcholine receptors in response to electric fields. In contrast to concanavalin A binding sites, which behave in a manner explained by electromigration and diffusion, acetylcholine receptors form stabile aggregates at the cathode-facing cell pole. Interestingly, receptor aggregation continues there after termination of the field. This post-field receptor aggregation is consistent with the action of a specific diffusion-trap mechanism mediated by adhesion or cohesion events on the cell surface. The simplest hypothesis for the triggering of receptor aggregation is that receptors are accumulated in response to the electric field and, once past a threshold density, autoaggregation is initiated. This model was tested by examining field-induced distributions of receptors following enzymatic treatment to reverse the electromigration of receptors. After neuraminidase treatment, and exposure to electric fields, receptor distributions were consistent with reversed electromigration (towards the anodal cell pole) but continued to show receptor aggregation at the cathodal cell pole. We conclude that the diffusion-trapping of acetylcholine receptors requires the participation of non-receptor molecules. In addition to providing a useful experimental tool in these studies, electric fields may play a role in receptor clustering at the neuromuscular junction.

Acetylcholine receptors and concanavalin A-binding sites on cultured Xenopus muscle cells: electrophoresis, diffusion, and aggregation.

Using digitally analyzed fluorescence videomicroscopy, we have examined the behavior of acetylcholine receptors and concanavalin A binding sites in response to externally applied electric fields. The distributions of these molecules on cultured Xenopus myoballs were used to test a simple model which assumes that electrophoresis and diffusion are the only important processes involved. The model describes the distribution of concanavalin A sites quite well over a fourfold range of electric field strengths; the results suggest an average diffusion constant of approximately 2.3 X 10(-9) cm2/s. At higher electric field strengths, the asymmetry seen is substantially less than that predicted by the model. Acetylcholine receptors subjected to electric fields show distributions substantially different from those predicted on the basis of simple electrophoresis and diffusion, and evidence a marked tendency to aggregate. Our results suggest that this aggregation is due to lateral migration of surface acetylcholine receptors, and is dependent on surface interactions, rather than the rearrangement of microfilaments or microtubules. The data are consistent with a diffusion-trap mechanism of receptor aggregation, and suggest that the event triggering receptor localization is a local increase in the concentration of acetylcholine receptors, or the electrophoretic concentration of some other molecular species. These observations suggest that, whatever mechanism(s) trigger initial clustering events in vivo, the accumulation of acetylcholine receptors can be substantially enhanced by passive, diffusion-mediated aggregation.

Mechanism for acetylcholine receptor localization at nerve-muscle synapse

This paper summarizes the hypothesis proposed to explain the mechanism for AChR localization at the neuromuscular synapse. Two theories have been proposed to explain the neuronal control of extrajunctional AChR. One theory claimed that motoneurons decreased the ACh sensitivity of the extrajunctional membrane through neurotrophic influence. However, direct electrical stimulation of denervated muscles resulted in a decrease of extrajunctional ACh sensitivity, supporting the other hypothesis that loss of extrajunctional AChR of the innervated muscle is directly related to muscle activity per se. AChR clusters (high density of AChR) at the neuro-muscular junction were supposed to result from the association of nerves with preexisting AChR clusters. However, Xenopus nerve-muscle cocultures clearly demonstrated that AChR clusters at the neuromuscular junction were formed after the nerve came in contact with the muscle membrane. Two hypothesis are proposed for nerve-induced formation of AChR clusters. Preferential insertion of AChR into the end-plate was suggested by the finding that AChR messenger RNA was more abundant near to than far from the end-plate in adult muscle fibers. On the other hand, in cultured and embryonic muscles, AChR clusters were formed at nerve-muscle junctions through receptor redistribution which was mediated by the passive diffusion-trap mechanism.

Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures

The formation of acetylcholine receptor (AChR) clusters at the neuromuscular junction was investigated by observing the sequential changes in AChR cluster distribution on cultured Xenopus muscle cells. AChRs were labeled with tetramethylrhodamine-conjugated α-bungarotoxin (TMR-αBT). Before innervation AChRs were distributed over the entire surface of muscle cells with occasional spots of high density (hot spots). When the nerve contacted the muscle cell, the large existing hot spots disappeared and small AChR clusters (less than 1 μm in diameter) initially emerged from the background along the area of nerve contact. They grew in size, increased in number, and fused to form larger clusters over a period of 1 or 2 days. Receptor clusters did not migrate as a whole as observed during “cap” formation in B lymphocytes. The rate of recruitment of AChRs at the nerve-muscle junction varied from less than 50 binding sites to 1000 sites/hr for αBT. In this study the diffusion-trap mechanism was tested for the nerve-induced receptor accumulation. The diffusion coefficient of diffusely distributed AChRs was measured using the fluorescence photobleaching recovery method and found to be 2.45 × 10 −10 cm 2/sec at 22°C. There was no significant difference in these values among the muscle cells cultured without nerve, the non-nerve-contacted muscle cells in nerve-muscle cultures, and the nerve-contacted muscle cells. It was found that the diffusion of receptors in the membrane is not rate-limiting for AChR accumulation.

Rapid lateral diffusion of functional A Ch receptors in embryonic muscle cell membrane.

During synaptogenesis of the skeletal neuromuscular junction, the acetylcholine (ACh) receptors dispersed over the surface of the embryonic muscle become highly concentrated at the subsynaptic membrane1,2. The mechanism by which a nerve induces such a modulation of ACh receptor topography is unknown. Of various possibilities3, the simplest explanation is that the ACh receptors, freely diffusing in the plane of the muscle membrane, are trapped at the site of nerve contact4,5. This ‘diffusion-trap’ mechanism is plausible only if the diffusion distance covered by the ACh receptors within its lifetime on the cell surface is at least comparable with the dimension of the embryonic muscle fibre (see also discussion in ref. 1). Photo-bleaching studies had suggested that this was not the case. In the present study, an electrophysiological method was used to measure the lateral diffusion of the dispersed, functional ACh receptors in the plasma membrane of cultured Xenopus embryonic muscle cells. The results suggest that these ACh receptors undergo rapid lateral diffusion with a diffusion coefficient of 2.6 × 10−9 cm2 s−1 at 22 °C. This rapid diffusion of the dispersed ACh receptors strongly supports a passive diffusion-trap mechanism for the localization of ACh receptors at the site of nerve–muscle contact.