Time-Resolved Measurement of State-Specific P2X<sub>2</sub>Ion Channel Cytosolic Gating Motions

Fisher, James; Girdler, Gemma; Khakh, Baljit S.

doi:10.1523/jneurosci.3250-04.2004

Cited by 53 publications

(90 citation statements)

References 54 publications

(108 reference statements)

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“…We determined whether Panx-1 channels contribute to P2X 2 ion permeability increases and YOPRO1 uptake, which reflect the I 2 state (2,3,5,9,20). We chose to work on P2X 2 because of the wealth of available structure-function data (7, 8) and because P2X 2 receptor activation does not lead to cell death and downstream effects like those reported for P2X 7 (7).…”

Section: Resultsmentioning

confidence: 99%

“…This study concerns how P2X 2 receptors open to a high permeability open state, called I 2 (2)(3)(4)(5)(6). Upon binding ATP, P2X receptors undergo millisecond time-scale transitions that lead to the open I 1 state (Fig.…”

mentioning

confidence: 99%

“…1 A). The second model posits that the I 2 state is an intrinsic property of P2X receptors involving slow conformational changes that allow organic cations to flow (2)(3)(4)(5)(6)19). We call this the ''gating model'' (Fig.…”

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confidence: 99%

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Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Chaumont

Khakh

2008

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

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ATP-gated P2X receptors display ion permeability increases within seconds of receptor activation as the channels enter the I 2 state, which is permeable to organic cations and dye molecules. The mechanisms underlying this important behavior are not completely understood. In one model, the I 2 state is thought to be due to opening of Pannexin-1 (Panx-1) channels, and, in the second, it is thought to be an intrinsic P2X property. We tested both models by measuring ion and dye permeability and used a patch-clamp coordinated spectroscopy approach to measure conformational changes. Our data show that Panx-1 channels make no detectable contribution to the P2X 2 receptor I2 state. However, P2X 2 receptors display permeability dynamics, which are correlated with conformational changes in the cytosolic domain remote from the selectivity filter itself. Finally, the data illustrate the utility of a new approach, using tetracysteine tags and biarsenical fluorophores to measure site-specific conformational changes in membrane proteins. (Fig. 1A), which is permeable to cations, such as Na ϩ and Ca 2ϩ (7,8). In P2X 2 , P2X 4 , and P2X 7 receptors after seconds of activation by ATP, the channel undergoes additional seconds time-scale changes that increase permeability to organic cations, such as N-methyl-D-glucamine (NMDG) ϩ , and dyes, such as YOPRO1 (2,(4)(5)(6)9). This second open state is referred to as I 2 (Fig. 1 A). The first reports of the I 2 state permeable to large molecules go back 30 years, when ATP was shown to permeabilize cells (10). The mechanisms that underlie opening to the I 2 state remain unclear, even though it is a trigger for a variety of pathophysiological processes, including blebbing, microvesicle shedding, release of signaling molecules, permeablization, and cell death (7,11). In addition to the obvious relevance to ATP signaling, a better understanding of the I 2 state would also advance our understanding of the diversity of mechanisms used by ion channels. This is because ion permeability and/or selectivity filter dynamics are not unique to P2X but also occur for other channels (12, 13). In particular, the organic cation and dye permeable I 2 state of TRP channels (14, 15) shares similarity to P2X 2 receptors. A key open question is how the organic cation and dye permeable I 2 state arises.Two models exist to explain the I 2 state for P2X receptors. The first model proposes that P2X 7 -mediated dye uptake (16, 17) occurs via Pannexin-1 (Panx-1) channels, leading to the suggestion that the I 2 state is due to Panx-1 channels rather than P2X (11,18). In this study, we call this the ''Panx-1 model'' (Fig. 1 A). The second model posits that the I 2 state is an intrinsic property of P2X receptors involving slow conformational changes that allow organic cations to flow (2-6, 19). We call this the ''gating model'' (Fig. 1 A). We tested both models under a common set of recording conditions. We also used a patch-clamp coordinated imaging approach to directly measure protein conformational changes. Results...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Chaumont

Khakh

2008

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

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“…Physiological saline comprised the following: 125 mM NaCl/5 mM KCl/1 mM MgCl 2 /10 mM D-glucose/2 mM CaCl 2 /25 mM Hepes. Cells were imaged and loaded with 5 M Fluo-3/AM or fura-2/AM (Molecular Probes, Invitrogen, Paisley, U.K.) as described (33) (49). Calibration of the Ca 2ϩ signal was achieved by permeabilizing the cells, determining the 340/380 ratio minimum (R min ) and maximum (R max ), and calibrating by using algorithms (50) Exocytosis Imaging.…”

Section: Methodsmentioning

confidence: 99%

“…Mixed hippocampal neuron-astrocyte cultures were transfected with 1-2 g of synaptopHluorin (18,19) cDNA by using Effectene (Qiagen). Twenty-four hours later, the cells were washed with buffer and imaged with evanescent wave microscopy as described (31,49). The camera gain (Princeton Instruments cooled I-PentaMAX camera; Roper Scientific, Trenton, NJ) was adjusted for each astrocyte to provide the best signal to noise images, and so comparisons between astrocytes can only be reported as ⌬F/F.…”

Section: Methodsmentioning

confidence: 99%

Two forms of single-vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy

Bowser

Khakh²

2007

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

143

168

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Transmitters such as glutamate and ATP are released from brain astrocytes. Several pathways for their release have been proposed, including exocytosis. In the present study we sought to measure exocytosis from astrocytes with single vesicle imaging methods using synaptopHlourin (SpH) as an optical reporter. We imaged single SpH-laden vesicles with total internal reflection fluorescence (TIRF) microscopy. We observed spontaneous, as well as evoked, single-vesicle exocytosis events. Analysis of the kinetics and spatial spread associated with these events indicated two discernible forms of single vesicle exocytosis. One form, constituting Ϸ40% of the spontaneous events, was akin to kiss-and-run vesicle fusion and captured a mobile proton buffer from the extracellular medium. The other form seems to represent full vesicle fusion, constitutes Ϸ60% of the spontaneous events, and is associated with complete mixing of the vesicle and plasma membranes. Activation of calcium-mobilizing receptors on the astrocyte surface selected between the different forms of exocytosis. These data provide evidence for two forms of simultaneously occurring singlevesicle exocytosis events in astrocytes, and also suggest that SpH imaging and TIRF microscopy is useful to study the mechanisms of astrocyte transmitter release.imaging ͉ calcium ͉ ATP

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Structure and Physiological Role of Ion Channels Studied by Fluorescence Spectroscopy

Corry

Cranfield

Martinac

2000

Encyclopedia of Analytical Chemistry

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Fluorescence spectroscopy has, over the last two decades, been frequently used for studies of biological cells and their molecular components. In combination with molecular biological methods that allow introduction of fluorescent labeling in vivo and in vitro , fluorescence spectroscopy methods, such as Förster resonance energy transfer ( FRET ), have made membrane proteins accessible to studies of their molecular structure and dynamics. In this article, we describe a variety of fluorescence spectroscopy techniques and focus on their use in the studies of the physiological role ion channels play, and the conformational rearrangements involved in the gating of ion channels, whose function as gated membrane pores underlies numerous cellular processes essential for the survival of living cells and organisms.

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Time-Resolved Measurement of State-Specific P2X₂Ion Channel Cytosolic Gating Motions

Cited by 53 publications

References 54 publications

Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Two forms of single-vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy

Structure and Physiological Role of Ion Channels Studied by Fluorescence Spectroscopy

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Time-Resolved Measurement of State-Specific P2X2Ion Channel Cytosolic Gating Motions

Cited by 53 publications

References 54 publications

Patch–clamp coordinated spectroscopy shows P2X 2 receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Patch–clamp coordinated spectroscopy shows P2X 2 receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Two forms of single-vesicle astrocyte exocytosis imaged with total internal reflection fluorescence microscopy

Structure and Physiological Role of Ion Channels Studied by Fluorescence Spectroscopy

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Time-Resolved Measurement of State-Specific P2X₂Ion Channel Cytosolic Gating Motions

Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

Patch–clamp coordinated spectroscopy shows P2X ₂ receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels