Secondary Structure and Gating Rearrangements of Transmembrane Segments in Rat P2X4 Receptor Channels

Silberberg, Shai D.; Chang, Tsun-Hsu; Swartz, Kenton J.

doi:10.1085/jgp.200409221

Cited by 67 publications

(94 citation statements)

References 69 publications

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“…The internal pore in our open-state model of the zfP2X4 receptor is the narrowest region of the pore, and it contains the most extensive intersubunit interactions, providing an initial picture of the structure of this important region of P2X receptor channels. Formation of intersubunit interfaces with the internal region of TM2 is consistent with observations that this region of TM2 is particularly sensitive to mutations (6,(27)(28)(29), and the model is fully compatible with metal bridges that have been described for the TM domain of rP2X2 receptor channels. These bridges include an intersubunit metal bridge formed at the threefold axis by V343C (Fig.…”

Section: Discussionsupporting

confidence: 86%

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

Heymann

Dai

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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P2X receptor channels open in response to the binding of extracellular ATP, a property that is essential for purinergic sensory signaling. Apo and ATP-bound X-ray structures of the detergentsolubilized zebrafish P2X4 receptor provide a blueprint for receptor mechanisms but unexpectedly showed large crevices between subunits within the transmembrane (TM) domain of the ATPbound structure. Here we investigate both intersubunit and intrasubunit interactions between TM helices of P2X receptors in membranes using both computational and functional approaches. Our results suggest that intersubunit crevices found in the TM domain of the ATP-bound crystal structure are not present in membraneembedded receptors but substantiate helix interactions within individual subunits and identify a hot spot at the internal end of the pore where both the gating and permeation properties of P2X receptors can be tuned. We propose a model for the structure of the open state that has stabilizing intersubunit interactions and that is compatible with available structural constraints from functional channels in membrane environments.pore-opening mechanism | transmembrane intersubunit crevices P 2X receptor channels are a family of trimeric cation-selective channels that are activated by extracellular ATP (1, 2). These ligand-gated ion channels are expressed in many tissues, including the central and peripheral nervous systems and the immune system, where they play a range of important roles in sensory signaling and inflammation (1, 3).Recent X-ray structures of the zebrafish P2X4 (zfP2X4) receptor in apo and ATP-bound forms (Protein Data Bank ID codes 4DW0 and 4DW1, respectively) have revealed the molecular design of these proteins (2, 4, 5) and have provided valuable information on how ATP binding triggers the opening of the transmembrane (TM) pore ( Fig. 1 A and B). ATP binds to a cleft between subunits within the large extracellular domain, inducing cleft closure and an accompanying lateral flexing of the β-sheet connecting the extracellular domain to the TM domain (5). The apo structure of the zfP2X4 receptor reveals that the TM1 helix is positioned peripheral to the TM2 helix and that the TM2 helix occludes the pore at the central axis within the outer half of the membrane (4, 5). In accord with this structure, accessibility studies show that the TM2 helix lines the aqueous pore and that the residues forming the gate are positioned within the occlusion in the apo structure (6-8). Lateral fenestrations within the extracellular domain provide a path for ions to enter and exit the extracellular vestibule positioned above this TM2 occlusion, and these fenestrations are thought to change conformation in response to ATP binding (9, 10). The ATP-bound structure shows that pore opening involves widening of the extracellular vestibule and an iris-like expansion of the pore (5). Intersubunit interactions within the TM domain in the apo structure are limited to the gate region of TM2 (5), and thus the pore expansion observed in the ATP-bound struc...

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Section: Discussionsupporting

confidence: 86%

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

Heymann

Dai

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Overexpression of wild-type (WT) P2X 4 tagged with EGFP had no effect on the frequency of FACE after stimulation with 100 μM ATP. Overexpression of the dominant-negative (DN) mutant P2X 4 (C353W)-EGFP, which has been reported to dramatically decrease Ca 2+ currents in P2X 4 receptor units when coexpressed with WT P2X 4 (45), significantly reduced the percentage of fusions followed by localized Ca 2+ entry (Fig. 3A).…”

Section: Resultsmentioning

confidence: 94%

Fusion-activated Ca ²⁺ entry via vesicular P2X ₄ receptors promotes fusion pore opening and exocytotic content release in pneumocytes

Miklavc

Mair

Wittekindt

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

136

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Ca2+ is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca 2+ concentration ([Ca 2+ ]) c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca 2+ channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca 2+ entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a "fusion-activated" Ca 2+ entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200-500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca 2+ channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X 4 receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X 4 receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X 4 . Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca 2+ influx via vesicular Ca 2+ channels regulates fusion pore expansion and vesicle content release.egulated secretion is a fundamental cellular process in many different types of eukaryotic cells, with Ca 2+ -triggered exocytosis being the key element (1-4). Multiple Ca 2+ -dependent steps have been elucidated that ultimately lead to fusion of exocytic vesicles with the plasma membrane, resulting in formation of an aqueous channel, the fusion pore, through which vesicle contents are released (5-8). Although the molecular composition of the fusion pore remains elusive, there is a general acceptance that fusion pores are not merely passive structures, but that their opening and closure are highly regulated and control, or even fine-tune, vesicle content secretion (9-14). Voltage-gated Ca 2+ channels are not present (25). After LB fusion with the plasma membrane, surfactant, a water-insoluble bulky complex, largely remains entrapped within the fused vesicles (26) in which the fusion pores behave as regulated valves or mechanical barriers for release (16,27). As a result, in vitro full content release can be delayed for minutes up to hours (28).We recently reported a "fusion-activated" Ca 2+ entry (FACE) mechanism as a phenomenon in the postfusion phase of surfactant secretion (29). Given that this Ca 2+ signal occasionally spreads throughout the cell, we speculated that it might be important for triggering...

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“…The low potency of BzATP relative to ATP strongly argues against homomeric P2X7 receptors (Surprenant and North, 2009). That said, the ATP EC 50 for native C8-B4 cells P2X4 receptors at 33 µM was somewhat higher than for rodent P2X4 receptors expressed in HEK cells that have been reported to be between 2 and 12 µM (Jones et al, 2000;Silberberg et al, 2005). This is reminiscent of differences between natively and heterologously expressed P2X2 receptors (Khakh et al, 2001).…”

Section: Properties Of Atp-evoked Currents In Activated C8-b4 Cellsmentioning

confidence: 99%

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

Toulmé¹,

Garcia²,

Samways³

et al. 2010

J Cell Biol

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We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2-6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by 3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking.

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Secondary Structure and Gating Rearrangements of Transmembrane Segments in Rat P2X4 Receptor Channels

Cited by 67 publications

References 69 publications

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

Fusion-activated Ca ²⁺ entry via vesicular P2X ₄ receptors promotes fusion pore opening and exocytotic content release in pneumocytes

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

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Secondary Structure and Gating Rearrangements of Transmembrane Segments in Rat P2X4 Receptor Channels

Cited by 67 publications

References 69 publications

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels

Fusion-activated Ca 2+ entry via vesicular P2X 4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

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Fusion-activated Ca ²⁺ entry via vesicular P2X ₄ receptors promotes fusion pore opening and exocytotic content release in pneumocytes