Featured ArticleCellular/Molecular

An Angstrom Scale Interaction between Plasma Membrane ATP-Gated P2X2 and α4β2 Nicotinic Channels Measured with Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Microscopy

Baljit S. Khakh, James A. Fisher, Raad Nashmi, David N. Bowser and Henry A. Lester
Journal of Neuroscience 20 July 2005, 25 (29) 6911-6920; DOI: https://doi.org/10.1523/JNEUROSCI.0561-05.2005

Abstract

Structurally distinct nicotinic and P2X channels interact functionally, such that coactivation results in cross-inhibition of one or both channel types. It is hypothesized, but not yet proven, that nicotinic and P2X channels interact at the plasma membrane. Here, we show that plasma membrane α4β2 nicotinic and P2X2 channels form a molecular scale partnership and also influence each other when coactivated, resulting in nonadditive cross-inhibitory responses. Total internal reflection fluorescence and fluorescence resonance energy transfer microscopy between fluorescently labeled P2X2 and α4β2 nicotinic channels demonstrated close spatial arrangement of the channels in human embryonic kidney cells and in hippocampal neuron membranes. The data suggest that P2X2 and α4β2 channels may form a dimer, with the channels ∼80 Å apart. The measurements also show that P2X2 subunits interact specifically and robustly with the β2 subunits in α4β2 channels. The data provide direct evidence for the close spatial apposition of full-length P2X2 and α4β2 channels within 100 nm of the plasma membrane of living cells.

Introduction

Transmitter-gated ion channels are found in neurons and synapses, where they bind neurotransmitter and convert this presynaptic chemical signal into a postsynaptic signaling event (Hille, 2001). Mammalian transmitter-gated ion channels consist of three families: Cys-loop channels for ACh, GABA, glycine, and serotonin; glutamate-gated channels (kainate, AMPA, and NMDA); and the ATP-gated P2X channels (Green et al., 1998; Khakh, 2001; Lester et al., 2004), of which seven subunits are known (P2X1-P2X7). There is little sequence, topological or structural similarity between members of the Cys-loop, glutamate-gated, and P2X channel family members (North, 1996a,b), leading to the assumption that they act independently in neurons and synapses.

Motivated by pioneering work on neurons showing that P2X and nicotinic channels may not act independently (Nakazawa, 1994; Barajas-Lopez et al., 1998; Searl et al., 1998; Zhou and Galligan, 1998), we suggested that neuronal nicotinic and P2X channels undergo cross-inhibitory functional interactions because of conformational coupling akin to conformational spread (Khakh et al., 2000). Conformational spread has been proposed as a general mechanism for signal integration by cells (Bray and Duke, 2004): conformational changes in one protein are relayed to its neighbors because of close spatial interactions. Cross-inhibition also occurs for P2X, nicotinic, GABA, and 5-HT3 subunits in heterologous expression studies as well as for natively expressed channels in neurons and synapses (Barajas-Lopez et al., 1998; Searl and Silinsky, 1998; Searl et al., 1998; Zhou and Galligan, 1998; Khakh et al., 2000; Sokolova et al., 2001b; Barajas-Lopez et al., 2002; Boue-Grabot et al., 2003, 2004a,b). One functional measure of cross-inhibition between ATP-gated P2X and ACh-gated nicotinic channels is that coapplication of ATP and ACh to cells expressing these channels evokes responses smaller than the sum of the individual ATP and ACh-evoked currents (Searl and Silinsky, 1998). This lack of summation implies that, when P2X and nicotinic channels are simultaneously activated, fewer channels open than expected if one assumes that the two channel populations function independently. The underlying mechanisms responsible for cross-inhibition between P2X2 and nicotinic channels remain incompletely understood.

If conformational spread is the cause of cross-inhibition between P2X2 and nicotinic channels, a close spatial arrangement between the channels in the plasma membrane is expected (Bray and Duke, 2004) but has remained unproven. We took advantage of recent data demonstrating that both P2X2 and α4β2 channels can be innocuously labeled with GFP-based fluorophores in their cytosolic domains (Khakh et al., 2001; Nashmi et al., 2003; Fisher et al., 2004). We first asked whether channels formed by these brain-expressed subunits (P2X2, α4, β2) (Kanjhan et al., 1999; Labarca et al., 2001; Fonck et al., 2003) show cross-inhibition similar to that demonstrated for channels found in the peripheral nervous system, and we then used fluorescence resonance energy transfer (FRET) microscopy to probe interactions between P2X2 and α4β2 channels in the plasma membrane of single cells.

Materials and Methods

Molecular biology. cDNAs for wild-type (wt) P2X2, α4, and β2 subunits, as well as cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-labeled P2X2 (P2X2-C, P2X2-Y), α4 subunits (α4-Y, α4-C), and β2 subunits (β2-C, β2-Y) were used (where C is CFP, and Y is YFP). These were all available from previous work (Nashmi et al., 2003; Fisher et al., 2004). For the generation of P2X6-C and P2X6-Y, the starting vectors were P2X2-C and P2X2-Y. In these vectors, HindIII (5′) and XhoI (3′) digestion yielded P2X2. By PCR (PCR Core kit; Qiagen, Crawley, UK) the P2X6 coding region was amplified (no stop codon) with noncoding 5′ HindIII and in frame 3′ XhoI sites, using plasmids encoding P2X6 as template (Khakh et al., 1999). These PCR products were cloned into HindIII and XhoI sites of P2X2-C and P2X2-Y to generate P2X6-C and P2X6-Y, respectively. All constructs were sequenced (Lark Technologies, Saffron Walden, UK or MRC Gene Service, Cambridge, UK).

Human embryonic kidney cell culture and transfection. Human embryonic kidney 293 (HEK 293) cells (obtained from American Type Culture Collection via LGC Promochem, Teddington, UK) were maintained in 75 cm2 cell culture flasks (Corning; Fisher Scientific, Loughborough, UK) in DMEM/Ham's F-12 media with Glutamax (Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were grown in a humidified atmosphere of 95% air/5% CO2 at 37°C in a cell culture incubator. The cells were split 1 in 10 when confluence reached 60-90%, which was generally every 3-4 d. Cells were prepared for transfection by plating onto six-well plates at the time of splitting, 3-4 d before transfection. They were transfected at 60-90% confluence. For transient expression in HEK 293 cells, we used 0.5-1 μg of plasmid cDNA and the Effectene transfection reagent (Qiagen) for each well of a six-well plate. The manufacturer's instructions were followed, with 4 μl of enhancer and 10 μl of Effectene used for each transfection. The transfection efficiency was 40-60%. For the functional measurements of cross-inhibition between P2X2 and α4β2 channels, we used a previously available stable cell line expressing α4β2 channels (Egan and Khakh, 2004), which has been described in detail previously (Chavez-Noriega et al., 2000), and transfected subunit cDNAs using procedures described above. All recordings were made 1-2 d after transfection, as described below. For FRET experiments, we used wild-type HEK cells and transfected cDNAs for wt α4, α4-Y, α4-C, wt β2, β2-Y, β2-C, P2X2-Y, and P2X2-C subunits in all combinations, as appropriate (see Results and Table 1). Using this approach, we tested all possible partnerships between α4β2 and P2X2 channels with FRET (see Results) in a cell type otherwise devoid of α4β2 or P2X2 channels, thus avoiding the complication of nontagged subunits to FRET.

View this table:
Table 1.

Average data for FRET efficiency and a measure of channels within 100 Å of each other when expressed in HEK cells and hippocampal neurons

Hippocampal neuron culture and transfection. Typically five rat pups at embryonic day 19 (E19) were used for hippocampal cultures. The mother was killed following Home Office (London, UK) and local procedures, and the embryos were removed to ice-cold dissection medium comprising Earle's buffered salt solution (with no Ca2+ or Mg2+ and no phenol red; Invitrogen), HEPES (10 mm, pH 7.4; Sigma, Poole, UK), and penicillin/streptomycin (1:100 dilution; Invitrogen). Hippocampi were dissected in Petri dishes filled with ice-cold medium. The dissected hippocampi (in medium, on ice) were cut in half, transferred to a 15 ml tube with 10 U/ml papain PAP2 powder [Worthington Biochemical (Lakewood, NJ) PAPAIN-022], and incubated for 15 min at 37°C. When the pieces of tissue had settled, digestion solution was removed and 10 ml of cold culture medium was added. This step was repeated, and the pieces were triturated five to six times in a 2 ml volume of ice-cold culture medium with paired flame-polished pipettes of progressively smaller bores. A hemacytometer was used to quantify yield (typically 2.5 million/ml), and ∼200,000 neurons were used for plating onto each coverslip. The coverslips were precoated with poly-d-lysine (50 μg/ml; Sigma) and then overnight with 150 μl of 20 μg/ml laminin (Sigma) in sterile dissection medium. Three hours after plating, the cells were fed with 2 ml of prewarmed culture medium and fed again 1 d after plating by substituting 1 ml of fresh prewarmed medium for 1 ml of existing medium. Cells were then fed every 4-5 d and used for experiments after 14 d in vitro. The culture medium comprised MEM with no phenol red (Invitrogen), glucose (20 mm), penicillin/streptomycin (Invitrogen), Na pyruvate (2 mm; Sigma), HEPES (25 mm; Sigma), N2 supplement (1:100 dilution; Invitrogen), and heat-inactivated horse serum (10%; Invitrogen). Effectene transfection reagent was used to transfect 0.5-1.0 μg of the appropriate cDNA per well of a six-well plate, which contained 3-4 coverslips. The instructions of the manufacturer were followed, except that 8 μl of enhancer and 24 μl of Effectene were used per 1 μg of cDNA. Experiments were performed 1-2 d after transfection, when transfection was successful typically in 1-2% of cells.

Midbrain mesencephalic dopaminergic neuron cultures and transfection. Ventral mesencephalic neuronal cultures were prepared as described previously (Nashmi et al., 2003). Brain tissue containing primary mesencephalic neurons of the ventral tegmental area from E14 BDF1 mouse embryos (Shimoda et al., 1992) was incubated in plating medium (Neurobasal, 2% B27, 0.5 mm Glutamax, and 5% horse serum) with 1 mg/ml papain at 37°C for 20 min. The digested tissue was mechanically dispersed, laid on 4% bovine serum albumin (Fraction V; Sigma, St. Louis, MO) in PBS, centrifuged for 6 min at 300 × g and resuspended in plating medium. Cells (∼150,000) were plated onto 35 mm culture dishes with 10-mm-diameter glass coverslip bottoms (MatTek, Ashland, MA) that were coated with poly-dl-lysine (catalog #P9011; 1 mg/ml; Sigma, St. Louis, MO) dissolved in sterile water. To transfect one dish of neurons, 1 μg of each cDNA was mixed with 60 μg of Nupherin-neuron (Biomol Research Laboratories, Plymouth Meeting, PA) in 400 μl of Neurobasal medium without phenol red. Lipofectamine 2000 (3 μl; Invitrogen, Carlsbad, CA) was mixed in 400 μl of Neurobasal media. The two solutions were incubated for 15 min at room temperature, combined, and incubated for another 45 min at room temperature. The medium of the mesencephalic cultures was replaced with plating medium without added serum, and the transfection medium was added. The culture dishes were centrifuged at 100 × g for 5 min on a swinging bucket rotor and incubated for 30 min at 37°C. Then, the transfection mixture was removed and replaced with the original plating medium. Experiments were performed 2 d after transfection, when transfection was typically successful in 1-2% of cells.

Electrophysiology. HEK 293 cells were used for recordings 24-48 h after transfection, as described previously (Fisher et al., 2004). The cells were gently mechanically dispersed and plated onto glass coverslips 2-12 h before use. We included this step to ensure that the cells became spherical and therefore adequately space clamped during recordings (Egan et al., 1998). The extracellular recording solution comprised (in mm) 147 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose, pH 7.4, and the pipette solution comprised (in mm) 154 KCl (or CsCl), 11 EGTA, and 10 HEPES. Whole-cell voltage-clamp recordings were made with 5 MΩ borosilicate glass electrodes (World Precision Instruments, Stevenage, UK), using an Axopatch 1D amplifier controlled by a computer running pClamp 8.1 software via a Digidata 1322 interface (Molecular Devices, Union City, CA). Data were filtered at 0.5-2 kHz and digitized at 1-5 kHz. Agonists were applied for 0.2-4 s at 2-3 min intervals using triple-barreled theta-glass (Egan and Khakh, 2004) and a rapid solution changer system (Perfusion Fast-Step System SF-77; Warner Instruments, Hamden, CT). Voltage ramps (0.5 s duration; -120 to +60 mV) were applied at the peak response to each agonist.

Confocal FRET microscopy. Confocal FRET imaging of midbrain neurons has been described previously (Nashmi et al., 2003). Cultured and transfected mesencephalic neurons were visualized at room temperature in extracellular buffer comprising (in mm) 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 d-glucose, and 1 ascorbic acid, pH 7.4. Ascorbic acid was included to minimize damage to dendrites during photodestruction. A series of λ-stack x-y images were collected from an upright LSM 510 Meta laser-scanning confocal microscope using an Achroplan IR 63× 0.9 numerical aperture (NA) water-immersion objective (Zeiss, Oberkochen, Germany). Images were collected over a spectral range between 462.9 and 602 nm with bandwidths of 10.7 nm after excitation of CFP with the 458 nm laser line of an argon laser. Pinhole was 1.32 Airy units, and the Z resolution was ∼2.0 μm. Images were collected at a 12-bit intensity resolution over 512 × 512 pixels at a pixel dwell time of 12.8 μs. FRET was recorded by examining the dequenching of CFP after incremental photodestruction of YFP by the 514 nm argon laser line. λ Stacks were acquired at various time points before and after photodestruction of YFP. These time points included 0, 0.13, 0.26, 0.52, 1.05, 1.57, 2.09, and 6.81 min. We separated the fluorescence contribution of each fluorescent protein (CFP or YFP) using a linear unmixing algorithm based on the spectral signatures of YFP and CFP created from reference λ-stack images of cells expressing either soluble YFP or soluble CFP, respectively. These unmixed YFP and CFP images (Dickinson et al., 2001; Lansford et al., 2001) were analyzed for FRET efficiency. Background fluorescence for both CFP and YFP, determined as the mean fluorescence intensity from an area containing cells that did not express the constructs, was subtracted from the overall intensity. Mean fluorescence intensities of CFP and YFP were determined by tracing the outer perimeter of the cell, the neuronal soma, or dendrite with a region of interest. However, in the case of FRET calculated for cell membrane surface receptors, mean fluorescence intensity was calculated for only the outer ∼2 μm shell of fluorescence and did not include the cell interior.

Epifluorescence FRET microscopy. We used an Olympus (Southall, UK) BX50 microscope equipped with a Peltier-cooled (-15°C) half-inch interline transfer chip Imago CCD camera (640 × 480 pixels; each pixel, 9.9 × 9.9 μm), epifluorescence condenser (Olympus IX two-port flash/imaging condenser), control unit (containing ISC and PDC boards), and the Polychrome IV monochromator (all from TILL Photonics, Gräfelting, Germany). The light from the monochromator was led to the microscope through a quartz fiber light guide. The hardware was controlled by a personal computer, an appropriate frame grabber (TILL Photonics), and macros driven by TILLvisION version 3.3 software. The cells were viewed with a 40× water-immersion objective lens with a numerical aperture of 0.8 (Olympus). We used the following filters for acquiring CFP or YFP images (in nm; all from Glen Spectra, Stanmore, UK): CFP (dichroic, 455DRLP; emitter, 480AF30) and YFP (dichroic, 525DRLP; emitter, 545AF35). To photodestroy the YFP, we used 525 nm light from the monochromator, and a 525DRLP dichroic. FRET was determined as described below.

Total internal reflection fluorescence microscopy and FRET. Briefly, we used an Olympus IX70 microscope equipped with a Princeton Instruments (Trenton, NJ) cooled I-PentaMAX camera with High Blue Gen III Intensifier (Roper Scientific, Trenton, NJ). The control of excitation and image acquisition was achieved using MetaMorph software and drivers (Universal Imaging, Marlow, UK) and shutters, filter wheels, and Proscan II control box (Prior Scientific, Cambridge, UK). The beams of 488/515 nm Ar (150 mW) and 442 nm He-Cd (12.5 mW) lasers (Melles Griot, Carlsbad, CA) at <5% power (constant for all experiments reported) were combined and controlled with an IX2-COMB (Olympus), Uniblitz shutters (Prior Scientific) and acousto-optical tunable filter and controller (AA Optoelectronics, Les Chevreuse, France) and fed into a fiber optic (FV5-FUR; Olympus) for entry into the total internal reflection fluorescence microscopy (TIRF) condenser (IX2-RFAEVA-2; Olympus). Cells were plated onto glass-bottom Petri dishes (170 μm thick; Willco Wells, Amsterdam, The Netherlands) 24-48 h before imaging and were viewed with a 60× oil-immersion objective lens, NA 1.45 (Olympus). FRET was determined with donor dequenching. During donor dequenching experiments on the TIRF rig, we noted that significant photodestruction of YFP occurred within ∼10 s, versus many minutes for epifluorescence imaging (Fisher et al., 2004). This difference, likely reflecting the use of different microscopes (IX70 vs BX50), objective lenses (60×, NA 1.45 vs 40×, NA 0.8), and light sources (laser vs xenon lamp), implied that, for TIRF experiments, we would obtain only a single time point for donor dequenching (see below, Data analysis and Results). The camera gain was adjusted for maximum signal-to-noise for each cell and kept constant for all image acquisitions of a particular cell.

Data analysis. For donor dequenching experiments, the FRET efficiency (e) was calculated as follows: Math(1)

where IC-before is the donor fluorescence intensity before photodestruction, and IC-after is the intensity after photodestruction. The photodestruction of the YFP proceeds with a rate equivalent to the dequenching of the donor, and plotting the photorecovery versus photodestruction yields a straight line (see Fig. 1). We used such linear plots and extrapolated to 100% acceptor photodestruction to calculate the maximum donor dequenching for epifluorescence microscopy (see Fig. 1): e is given by the y-axis intercept. For TIRF microscopy, we estimated FRET e from a single time point when the acceptor was ∼85% destroyed and extrapolated to 100% destruction by constraining the linear regression to pass through the origin. This was the most appropriate tactic, because it was not possible to track time-dependent photodestruction with TIRF: on one hand, as noted, the bleaching occurred too rapidly with laser illumination (<0.5-1 min). In contrast, with epifluorescence illumination, bleaching typically took ∼30 min, allowing multiple evenly spaced time points to be collected accurately (see Results) (Fisher et al., 2004); however, because during TIRF only the plasma membrane within 100 nm of the glass-cell interface, the footprint, is illuminated (Steyer and Almers, 2001), it is necessary to destroy the acceptor fluorophore rapidly within this region and to measure donor dequenching before channels that are not within the evanescent field of illumination (and therefore neither destroyed or dequenched) can either diffuse or be inserted into the foot-print. Thus, the fact that for TIRF microscopy maximal photodestruction occurred rapidly was a fortuitous feature that enabled us to determine FRET from a single time point. In Results, we present a detailed comparison of FRET values determined for a single time point versus those calculated from multiple time points.

To approximate distances, we used the Förster equation: Math(2)

where Math(3)

e is the experimentally determined FRET efficiency, and R is the distance between donor and acceptor fluorophores (Michalet et al., 2003; Bunt and Wouters, 2004). R0 includes terms for the donor quantum efficiency (φD), the solvent refractive index (n), overlap of the donor emission and acceptor absorption spectra (JDA), and the orientation factor (κ2). R0 is the distance at which e is 50% for any given fluorophore pair. In the case of CFP and YFP, this is 49.2 Å (Patterson et al., 2000) or 50.4 Å (Tsien, 1998). We have no good reason to choose between these values and have taken the average and rounded to the nearest whole number to give a value of 50 Å (5 nm). Importantly, R0 contains a term for the fluorophore dipole orientation factor (κ2), which, at 2/3, assumes random tumbling of the fluorophores (Bunt and Wouters, 2004). This means that R0 and the distance measurements reported here assume that the fluorophores adopt all spherically random orientations. Given that we do not know the relative orientation of the fluorophores on P2X2 and nicotinic channels, assuming a κ2 value of 2/3 seems parsimonious and appropriate (Michalet et al., 2003; Bunt and Wouters, 2004) and is supported by empirical data for CFP and YFP fused to the cytosolic C termini of inwardly rectifying K+ channels (Riven et al., 2003), which have a membrane topology similar to P2X channels (North, 1996a). When interpreting FRET efficiency measurements in terms of distance, it is important to consider that, in the case of CFP and YFP, the fluorophore is buried in the center of a β-can 30 Å in width and 45 Å in length (Tsien, 1998). An additional consideration is that we do not know the absolute ratio of donors to acceptors during FRET at the single molecule level (see Discussion).

For the FRET experiments, we chose regions of interest (ROIs) post hoc (Fisher et al., 2004). Our criteria were that ROIs (1) were bright in both CFP and YFP images, (2) were located at the edge, presumably plasma membrane of the cell, (3) did not change or drift in intensity during the control period, and (4) remained in focus during the entire experiment.

For the electrophysiological experiments, we used analysis that has been described previously (Khakh et al., 2000). Briefly, we measured the currents activated by ATP (IATP), ACh (IACh), and ATP and ACh together (IATP/ACh). From these, we calculated the predicted current (IPredicted) and the occluded current (ILost) on a cell-by-cell basis, where Math(4) Math(5)

Thus, the percentage contribution of any one component such as IACh to the predicted current can be calculated as (IACh/IPredicted) × 100. Rectification index was calculated as the ratio of macroscopic slope conductances at voltages ±40 mV from the reversal potential. Reversal potentials were measured using two- or three-order polynomial fits to the current-voltage relationships and were taken as the values at which the fitted line first intersected the x-axis (see Fig. 1C, bottom). Electrophysiological analysis was performed with Clampfit (Molecular Devices) or Origin 6.1 (OriginLab, Northampton, MA), and all statistical tests were run in GraphPad Instat 3.0 (GraphPad Software, San Diego, CA). Image analysis was performed with ImageJ (http://rsb.info.nih.gov/ij/). Data in the text and graphs are shown as mean ± SEM from n determinations as indicated.

Results

Electrophysiological evidence for cross-inhibition between P2X2 and α4β2 channels

We applied ATP (100 μm) to activate P2X2 and ACh (100 μm) to activate nicotinic channels in HEK cells coexpressing wt P2X2 and α4β2 channels (Fig. 1A,B). We measured robust ATP- and ACh-evoked inward currents (at -60 mV: IATP at -1161 ± 311 pA and IACh at -522 ± 113 pA; n = 9) when either agonist was applied alone, but less than the predicted (IPredicted at -1635 ± 402 pA; n = 9) current when both ACh and ATP were coapplied (IATP/ACh) (Fig. 1B). The measured current (IATP/ACh at -1037 ± 265 pA; n = 9) on coapplication of ATP and ACh was consistently smaller than the predicted current by 39 ± 4% (ILost). This ILost percentage was thus approximately equal to the IACh percentage (35 ± 3%) and significantly smaller than the IATP percentage (70 ± 2%), where 100% equals IPredicted (see Materials and Methods, Data analysis). Interestingly, the rectification indices and reversal potentials of IATP/ACh (0.18 ± 0.03 and +6.6 ± 0.6 mV; n = 5) were similar to those of IATP (0.21 ± 0.02 and +7.5 ± 0.9 mV; n = 5) and significantly different from IACh (0.04 ± 0.02 and -5.4 ± 1.0 mV; n = 5) (Fig. 1C), as though few nicotinic channels opened after coapplication of ATP and ACh. Together, these data extend previous work on peripheral nervous system channels (see Introduction) to α4β2 nicotinic and P2X2 channels abundantly found in the brain (Kanjhan et al., 1999; Labarca et al., 2001; Fonck et al., 2003).

FRET between P2X2 and α4β2 channels in HEK 293 cells

We used the donor dequenching method (Michalet et al., 2003) to estimate FRET efficiency for P2X2 and α4β2 channels expressed in HEK 293 cells (Fig. 2A). If FRET occurs, one expects full removal (photodestruction) of the YFP acceptor to increase CFP fluorescence by a percentage amount that is equal to FRET e. We determined this value by plotting the photorecovery of CFP as a function of the photodestruction of YFP and estimated e when we extrapolated the data to 100% YFP photodestruction for a membrane ROI (Fig. 2B-E). Our values of e for P2X2-C/P2X2-Y and α4-Y/β2-C channels (Table 1, 28% and 25%, respectively) agree with previous work (Nashmi et al., 2003; Fisher et al., 2004). The 10% difference between α4-Y/β2-C (e = 25%) and α4-C/β2-Y (e = 34%) (Table 1, Fig. 2) may reflect a preferential α4:β2 stoichiometric ratio of 2:3 in the heteromeric nicotinic channels (Table 1).

Figure 1.
Figure 1.

Cross-inhibition between P2X2 and α4β2 channels expressed in HEK cells. A, Diagram of presently understood membrane topology and oligomeric state of nicotinic and P2X channels. N, N terminus; C, C terminus. B, Three examples of inward currents (-60 mV) evoked by sequential ∼5 s applications of ATP, ACh, and ATP/ACh from an HEK cell expressing P2X2 and α4β2 channels (for average data, see Results). The dashed line indicates the level of the peak predicted current if there was no cross-inhibition. C, Top, Representative current-voltage relationships of currents evoked by ATP, ACh, and ATP/ACh together (for average data, see Results). Bottom, Expanded plot near the reversal potential (Erev) for the current-voltage relationships shown in the top panel. The thin lines are the raw data, and the thick lines are two- or three-order polynomial fits to the data (see Materials and Methods, Data analysis). In all cases, agonists (100 μm) were applied at 2 min intervals.

Importantly, we also measured strong FRET between CFP- or YFP-labeled P2X2 and α4β2 channels under some conditions. FRET between P2X2 and α4β2 was strong when the cognate acceptor or donor fluorophore was on the β2 subunit (e = 23 and 26%) (Table 1, Fig. 2D) but was weak or absent when the acceptor (e = 10%) or donor (e = 0%) fluorophores were on the α4 subunit (Table 1, Fig. 2E). However, we know that the P2X2-β2 interaction does require assembled α4β2 channels, because no FRET was measured from cells expressing CFP-labeled P2X2 channels and YFP-labeled β2 subunits (e =∼0%; n = 9) (Table 1). These data demonstrate that (1) P2X2 and α4β2 channels were spatially close and that (2) FRET is stronger for P2X2 and β2 than for P2X2 and α4.

We next repeated the experiments with TIRF-FRET microscopy (Fisher et al., 2004). This method allows for the visualization of channels within ∼100 nm of the plasma membrane (Fig. 3A) in an area called the footprint (Steyer and Almers, 2001). We used donor dequenching to estimate FRET e. Figure 3B shows donor and acceptor images from cell footprints expressing P2X2-C/P2X2-Y, α4-C/β2-Y, and P2X2-C/α4β2-Y subunits before and after acceptor photodestruction. From images such as these, we calculated FRET e by extrapolating to 100% acceptor photodestruction (Fig. 4A) for equally sized footprints (Fig. 4B). In contrast to the experiments with epifluorescence microscopy (Fig. 2B,C), we could obtain only one time point, at ∼85% acceptor photodestruction (see Materials and Methods). FRET e was measured as 34 ± 3, 35 ± 4, 41 ± 5, and 22 ± 2% for P2X2-C/Y (n = 31), α4-Y/β2-C (n = 17), α4-C/β2-Y (n = 16), and P2X2-C/α4β2-Y channels (n = 22), respectively (Fig. 4B).

Figure 2.
Figure 2.

FRET between P2X2 and α4β2 channels in HEK cells. A, Donor (CFP) and acceptor (YFP) images of HEK cells expressing α4-C/β2-Y channels before and after photodestruction of the acceptor fluorophore. B, Linear plots of donor (CFP) photorecovery versus acceptor (YFP) photodestruction for channels formed by coexpressing P2X2-C and P2X2-Y channels. The y-axis intercept is a measure of FRET e (see Materials and Methods, Data analysis). C-E, As in B but for channels formed by coexpressing the indicated subunits. B-E, Linear plots for membrane ROI only, whereas Table 1 shows data from whole-cell ROI as well. Error bars represent SEM.

As discussed above (see Materials and Methods) it is necessary to photodestroy the acceptor rapidly for TIRF experiments, so as to minimize distortion by channels outside the evanescent field diffusing into the footprint. We could achieve this photodestruction readily within ∼10-30 s (see Materials and Methods) (Figs. 3B, 4A). However, with this approach, we were restricted to estimating FRET from a single time point (Fig. 4A), and we next sought to address the issue of whether measurements of FRET e from a single point are reliable compared with measurements of FRET e from several points. This was not possible for TIRF, because of the necessity for rapid acceptor photodestruction (see Materials and Methods), but was possible for epifluorescence experiments when the whole cell, rather than the footprint alone, was illuminated. For these experiments, we estimated FRET e from linear plots derived from a single point when the acceptor was ∼85% destroyed (as in the TIRF experiments) (Fig. 4A) and compared these values with FRET e estimates derived from linear plots such as those illustrated in Figure 2B using multiple points. We performed this analysis for six sets of constructs. Figure 4C shows near equality between FRET e estimates determined from a single point and those determined for the same constructs with multiple points. This indicates that the measurement of FRET e from a single point, when the acceptor is ∼85% photodestroyed, provides a reliable measure of true FRET e. Moreover, for our most complete data set (P2X2-C/P2X2-Y channels), the FRET e estimates determined with TIRF at a single time point (34 ± 3%; n = 31), epifluorescence with five time points (28 ± 8%; n = 7), epifluorescence with one time point (28 ± 8%; n = 7), or linear unmixing [33 ± 5%; n = 18 from Fisher et al. (2004)] were not significantly different. These data indicate that the technical necessity to estimate FRET from a single time point for the TIRF experiments (Fig. 4A) does not underestimate or overestimate FRET efficiency. These data and analysis give us confidence in the TIRF data presented in Figure 4A.

FRET between P2X2 and α4β2 channels in neurons

P2X2 and α4β2 channels are endogenously expressed in brain neurons (Kanjhan et al., 1999; Labarca et al., 2001; Fonck et al., 2003), and we next determined whether P2X2 and α4β2 channels undergo FRET when transfected into hippocampal neurons. We measured marked colocalization between P2X2-C/P2X2-Y, α4-C/β2-Y, and between P2X2-C and α4β2-Y channels in hippocampal neuron somata (supplemental Fig. 1, available at www.jneurosci.org as supplemental material), as well as strong FRET between P2X2 and α4β2 channels in hippocampal neuron membrane and in whole-cell ROIs (Table 1, Fig. 5A). This provides strong evidence that the FRET measured between P2X2 and α4β2 channels in HEK cells also occurs when the subunits are coexpressed in neurons that normally express these channel types (Kanjhan et al., 1999; Labarca et al., 2001; Fonck et al., 2003).

We repeated these experiments for ventral midbrain neurons, which also endogenously express P2X2 and α4β2 channels (Kanjhan et al., 1999; Labarca et al., 2001; Fonck et al., 2003). We measured strong colocalization between CFP- and YFP-labeled P2X2 channel subunits and between CFP- and YFP-labeled α4 and β2 subunits but weak colocalization between P2X2-C and α4β2-Y channels (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). Moreover, within midbrain neuron somata, we measured robust FRET for P2X2-C/P2X2-Y and α4-Y/β2-C channels (e = 38 ± 8%, n = 4; and 24 ± 6%, n = 3, respectively), but 0% FRET for P2X2-C and α4/β2-Y channels (Fig. 5C) (n = 5). These data suggest that there is no interaction between P2X2 and α4β2 channels in midbrain neuron somata, consistent with their distinctly weak colocalization (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). Data for dendrites followed a similar pattern: we measured strong FRET for dendritic P2X2-C/P2X2-Y and α4-Y/β2-C channels at 36 ± 5% (n = 7) and 27 ± 4% (n = 4), but 0% FRET for dendritic P2X2-C and α4/β2-Y channels (n = 7). The FRET differences between hippocampal and midbrain neurons extend previous functional studies suggesting that interactions between P2X and nicotinic channels may vary depending on the neuron type (Nakazawa, 1994; Rogers et al., 1997).

On the percentage of P2X2 and α4β2 channels within 100 Å of each other

Table 1 shows that FRET e was lower for whole cells compared with membrane ROI, in agreement with previous data (Nashmi et al., 2003; Fisher et al., 2004). If one assumes that channels in the plasma membrane display a maximal FRET e equal to that determined with our highest resolution experiments (TIRF microscopy) and also that unassembled subunits do not FRET because their separation greatly exceeds the Förster distance for CFP and YFP (5 nm; see Materials and Methods), the percentage of fluorescent channels that are within ∼100 Å (Nashmi et al., 2003) can be calculated as follows: Math(6)

where eTIRF is the FRET efficiency determined with TIRF, eROI is the value for FRET from an ROI (membrane or whole cell) from epifluorescence microscopy and enoise is the contribution of noise to the FRET measurements. We determined enoise from experiments with P2X6-C/P2X6-Y channels, because these are unassembled (Torres et al., 1999a) and not functionally expressed in the plasma membrane (Khakh et al., 1999). We calculated a FRET value of 3.5% for P2X6-C/P2X6-Y channels for whole-cell ROIs from the average data, because it was not possible to calculate FRET e for all of the single cells, some of which showed negligible photorecovery (Fig. 6). The low FRET value implies that P2X6 channels are expressed mainly as unassembled monomers, a suggestion consistent with previous biochemical experiments (Torres et al., 1999a). From the perspective of the present study, these data thus provide a measure of noise that may arise because of “nonspecific” FRET between subunits that occasionally pass within ∼80 Å of each other when coexpressed in cells (Fig. 6, Table 1). This value, 3.5%, is significantly lower than the FRET measurements for P2X2, α4β2, or P2X2/α4β2 channels (Table 1). Using equation 1, we calculated the percentage of fluorophore-labeled channels that were within ∼100 Å of one another for P2X2, α4β2, and P2X2/α4β2 channels in HEK cells and hippocampal neurons (Table 1). This value was generally higher for P2X2, α4β2, and P2X2/α4β2 channels in the membrane than for whole cells (Table 1), consistent with the suggestion that whole-cell ROIs contain contributions from the endoplasmic reticulum, which contain a proportion of unassembled channels (Nashmi et al., 2003; Fisher et al., 2004). We suggest that ∼30 and ∼100% of whole-cell and plasma-membrane channels formed by coexpressing P2X2 and α4β2 channels in HEK cells are dimers between these channel types, with an arrangement such that the P2X2 subunit is ≥80-100 Å from the α4 subunit, but within ∼60 Å of the β2 subunit (Fig. 7) (see Materials and Methods and Discussion).

Figure 3.
Figure 3.

FRET measurements for channels within 100 nm of the plasma membrane. A, Diagram of the principle of TIRF microscopy (Steyer and Almers, 2001). Channels within 100 nm of the glass-water interface are strongly optically excited, whereas channels within intracellular compartments and vesicles are not. This restricts FRET measurements to within ∼100 nm of the plasma membrane. B, Donor and acceptor images of HEK cell footprints expressing P2X2-C/P2X2-Y (top), α4-C/β2-Y (middle), and P2X2 and α4β2-Y (bottom) channels before and after photodestruction of the acceptor fluorophore. Note that clear intensity increases in the CFP images when the acceptor was photodestroyed.

Discussion

The main finding, summarized diagrammatically in Figure 7, is that P2X2 and α4β2 channels form a molecular scale partnership within 100 nm of the plasma membrane.

Functional and optical measures of an interaction between P2X2 and α4β2 channels

The electrophysiological data for P2X2 and α4β2 channels expressed in HEK cells demonstrate that these channels undergo functional interactions similar to those described previously for P2X2 and nicotinic, GABA, or 5-HT3 channels (Nakazawa, 1994; Barajas-Lopez et al., 1998; Searl and Silinsky, 1998; Searl et al., 1998; Zhou and Galligan, 1998; Khakh et al., 2000; Sokolova et al., 2001b; Barajas-Lopez et al., 2002; Boue-Grabot et al., 2003, 2004a,b). In future work, it will be important to test whether cross-inhibition also occurs for P2X and nicotinic channels in brain neuron nerve terminals, where they are often coexpressed (Kanjhan et al., 1999; MacDermott et al., 1999; Khakh and Henderson, 2000; Labarca et al., 2001; Fonck et al., 2003; Engelman and MacDermott, 2004). Indeed, there is evidence that some P2X-like channels colocalize and display cross-inhibition with nicotinic channels within cholinergic nerve terminals (Diaz-Hernandez et al., 2002, 2004).

Figure 4.
Figure 4.

FRET estimates from TIRF microscopy. A, Linear plots of donor (CFP) photorecovery versus acceptor (YFP) photodestruction for channels formed by coexpressing the indicated subunits. The y-axis intercept is a measure of FRET e (see Materials and Methods, Data analysis). B, Footprint areas for the cells shown in A. C, A comparison of FRET e values determined from a single point or for multiple points with epifluorescence microscopy (as in Fig. 2). The data for cytosolic CFP and YFP (CFP/YFP) and yellow cameleon 2.1 (YC2.1) determined with multiple points have been reported (Fisher et al., 2004). The data point for P2X6 has no error bars, because we could estimate FRET e only from the average data (see Results). Error bars represent SEM.

Figure 5.
Figure 5.

FRET for channels expressed in hippocampal and midbrain neurons. A, Representative donor and acceptor images of a hippocampal neuron expressing P2X2-C/P2X2-Y channels before and after photodestruction of the acceptor fluorophore. B, Linear plots of donor (CFP) photorecovery versus acceptor (YFP) photodestruction for channels formed by coexpressing P2X2-C and P2X2-Y channels in hippocampal neurons. The y-axis intercept is equal to FRET e. C, As in B but for subunits coexpressed in ventral midbrain neurons.

Interestingly, we found robust FRET between P2X2 and α4β2 channels in hippocampal neurons but not in midbrain neurons. Neuron-specific interactions also occur in the periphery: cross-inhibition occurred in sympathetic neurons from the celiac ganglion (Searl et al., 1998) but not in those from the superior cervical ganglion (Rogers et al., 1997). Perhaps there are neuron-specific cues that govern cross-inhibition, for example variable expression of interacting or targeting proteins.

We proposed previously that conformational spread may explain the ability of P2X2 and nicotinic channels to display cross-inhibition (Khakh et al., 2000). In the simplest cases, the proteins that display conformational spread are spatially close or linked (Bray and Duke, 2004) so that motions in a molecule are communicated to its neighbors. Conformational spread is well studied for bacterial chemotaxis and may also occur for receptors and channels (Bray and Duke, 2004). However, there have been no data on the spatial arrangement or possible physical association of P2X2 and nicotinic channels in the plasma membrane, a prerequisite for conformational spread. The present study provides FRET-based evidence for an angstrom scale interaction between full-length P2X2 and α4β2 nicotinic channels in the plasma membrane of living cells. Thus, we complement previous electrophysiological studies on cross-inhibition. The FRET measurements imply that P2X2 interacts more strongly with the β2 than with the α4 nicotinic receptor subunit and provide the percentage of channels that are within ∼100 Å of each other and also within 100 nm of the plasma membrane (Fig. 7B).

Molecular interpretations of FRET efficiency data

It is unlikely that the FRET data reported here arise from a non-specific phenomenon such as “overcrowding in the membrane.” First, FRET for P2X channels depends strongly on the subunit combination: P2X6 shows negligible FRET (0-3.5%), P2X1 displays intermediate FRET (19%), and P2X2 shows the highest FRET (30-35%) (this study and Fisher et al., 2004). Importantly, all these subunits express robustly in HEK cells (Torres et al., 1999a; North, 2002) and yet FRET varies by a factor of 10. This is inconsistent with simple overcrowding causing FRET in our experiments. Second, previous experiments show that FRET is specific to heteromeric α4β2 channels or to heteromeric GluCl channels and does not occur between nicotinic or GluCl subunits when they are expressed in the same cell (Nashmi et al., 2003), also arguing against overcrowding. Likewise, in this study, FRET for α4β2 channels varied depending on the α:β ratio, as expected of preferential donor/acceptor ratios (2:3 vs 3:2), arguing against overcrowding phenomena. Third, FRET between P2X2 and α4β2 channels was specific, occurring only between P2X2 and β2 subunits in α4β2 channels. If FRET occurred because of overcrowding, it would presumably not discriminate between α4 and β2 subunits in assembled α4β2 channels. Fourth, we measured no FRET in cells expressing P2X2 and β2 subunits alone. Fifth, direct imaging shows that P2X2 channels do not form clusters of over-crowded receptors but rather adopt a relatively uniform membrane distribution (Khakh and Egan, 2005). Sixth, both P2X2 and α4β2 channels are expressed at a density of <10 channels/μm2 (Khakh et al., 2001; Nashmi et al., 2003; Fisher et al., 2004; Fujiwara and Kubo, 2004; Khakh and Egan, 2005). In contrast, channels become crowded at plasma membrane densities >10,000 channels/μm2 (Hille, 2001). Seventh, FRET has been used for decades to measure protein-protein inter-actions; no substantive data or arguments question the validity of the approach because of nonspecific overcrowding in the membrane (Michalet et al., 2003; Bunt and Wouters, 2004). Future work may overcome the technical challenges of systematically varying the expression levels of P2X2 and α4β2 channels and determine the impact on FRET.

Figure 6.
Figure 6.

FRET after coexpression of CFP and YFP-labeled P2X6 subunits. A, Donor (CFP) and acceptor (YFP) images of two HEK cells expressing P2X6-C/P2X6-Y subunits before and after photodestruction of the acceptor fluorophore. Note the minimal dequenching of the donor. B, Linear plots of donor (CFP) photorecovery versus acceptor (YFP) photodestruction for channels formed by coexpressing P2X6 subunits. The y-axis intercept is equal to FRET e (see Materials and Methods, Data analysis).

The FRET efficiency values for P2X2 and α4β2 channels reported here agree with previous estimates (Nashmi et al., 2003; Fisher et al., 2004) and in the case of nicotinic channels with values expected from a 4.6 Å structural model (Miyazawa et al., 1999). Extrapolating from this model to α4β2 channels (Fig. 7A) suggests that the distance between the edges of any α4 subunit and its nearest or farthest β2 subunit would be ∼38 Å (a) or ∼45 Å (b), respectively. Note that the fluorophore is buried in a β-can ∼30 Å in diameter and ∼45 Å in length (Tsien, 1998). Assuming that the fluorescent protein domains line up with their long axes parallel, this implies that the surfaces of the two fluorescent protein domains are separated in α4β2 channels by ∼60 minus ∼30 Å (from two fluorophore radii) or ∼30 Å. We consider this probable because the five fluorophores likely stick out from the helices forming the α4β2 cytosolic domain rather than being buried within them, which we presume would be detrimental given the relatively small dimensions of, and available space within, the entire domain (Miyazawa et al., 1999). Because of the 1/R6 dependence, the estimate of ∼30 Å measures primarily the distance a between an α4 subunit and its nearest β2 (Fig. 7A). Finally, if the 2:3 α:β stoichiometry and arrangement of Figure 7 is correct, JDA is effectively doubled, and the value of ∼30 Å is an underestimate by ∼12%. Thus, the experiments suggest a distance estimate for α4β2 channels at ∼33 Å, comparable with 39-41 Å expected from a structural model of a related channel. Similar comparisons are not possible for P2X channels, because no structural models exist, but the FRET efficiency measurements agree roughly with expectations from structural models of the cytosolic domains of several ion channels (Chang et al., 1998; Miyazawa et al., 1999; Sokolova et al., 2001a; Higgins et al., 2002; Jiang et al., 2002; da Fonseca et al., 2003; Kuo et al., 2003).

The finding that FRET is ∼30-34% for P2X2 channels and ∼3.5% for P2X6 channels provides strong evidence that FRET efficiency for P2X channels is subunit specific. In previous data, P2X1 channels display intermediate FRET efficiency at ∼19% (Fisher et al., 2004). The difference for FRET between probes at the extreme C terminus of P2X2 and P2X1 channels may arise because P2X2 channels have a longer C terminus (120 residues) compared with P2X1 (47 residues); perhaps the P2X2 cytosolic domain resembles a phonograph needle, with the C terminus at the cytoplasmic tip (Fisher et al., 2004). The value of 3.5% represents an upper limit and probably measures noise in our experiments; FRET for P2X6 channels may not exist, consistent with electrophysiological (Le et al., 1998; Khakh et al., 1999), biochemical (Torres et al., 1999a), and atomic force microscopy studies (Barrera et al., 2005) demonstrating that P2X6 subunits do not oligomerize but remain as unassembled monomers. In contrast, P2X2 subunits apparently assemble as trimers (Aschrafi et al., 2004; Barrera et al., 2005). We are encouraged that FRET imaging experiments on several thousand channels in single living cells provide information consistent with biochemistry and atomic force microscopy on concentrated, isolated, and purified channels. FRET may take its place as a noninvasive approach to understand the molecular basis and dynamics of P2X channel assembly and trafficking (Torres et al., 1999a,b; Chaumont et al., 2004).

Figure 7.
Figure 7.

Diagrams summarizing FRET data. A, Summary of the FRET efficiency values determined with epifluorescence FRET imaging for membrane ROI. The blue and yellow dots represent CFP and YFP, respectively. The pentagon illustrates the shortest (a) and longest (b) straight line distances between α and β subunits. B, The diagrams illustrate the salient findings, namely that P2X2 is within 60 Å of the β2 subunit in α4β2 channels and >80 Å from the α4 subunit. By drawing the channels parallel to one another, we are not making any statements about their three-dimensional relationships to each other but rather schematically illustrating the key findings from FRET.

The interpretation of FRET efficiencies

Several uncertainties arise when one interprets FRET in terms of absolute distance. (1) We assume that the fluorophores adopt random orientations, so that the assumed orientation factor κ2 = 2/3 implies that R0 = 50 Å (see Materials and Methods). (2) In the case of trimeric P2X2 channels (Fig. 7A), it is important to note that 50% of the channels that undergo FRET have two donors and one acceptor, and 50% have one donor and two acceptors (Fisher et al., 2004). Fluorescence lifetime measurements might discriminate between these populations; however, in the absence of such experiments, our data likely represent an average of the FRET efficiencies from these different donor/acceptor ratios (Fig. 7A). (3) In α4β2 nicotinic channels, the fluorophore is embedded in M3-M4 intracellular loops of varying length, and presumably size, among subunits (α4, 271 aa; β2, 138 aa), which is expected to produce asymmetry. (4) α4β2 nicotinic channels are pentamers, with variable stoichiometry of α4 to β2 subunits rather than the fixed 2:3 α:β of Figure 7, and therefore unknown donor/acceptor ratios in our experiments (Zwart and Vijverberg, 1998; Nelson et al., 2003). These quantitative uncertainties do not vitiate the qualitative finding that FRET was robust and high for P2X2 and β2 subunits (Fig. 7A) and negligible between P2X2 and α4 subunits (Fig. 7A), indicating a strongly preferential order of subunits in a spatial arrangement encompassing P2X2 and α4β2 channels (Fig. 7B).

Summary and outlook

That structurally distinct transmitter-gated channels can form plasma membrane partnerships extends previous work on assembly of dopamine and somatostatin (Rocheville et al., 2000), adenosine (Gines et al., 2000), GABAA (Liu et al., 2000), and NMDA (Lee et al., 2002) receptors. Thus, functional interactions between structurally distinct receptors and channels may be a general mechanism. The finding that the P2X2 C terminus interacts specifically in living cells with the cytosolic domain of distinct subunits from the Cys-loop channel family is consistent with biochemical studies on interactions between P2X2 and GABA-gated ion channels (Boue-Grabot et al., 2004a,b). The close spatial apposition of P2X2 and α4β2 channel cytosolic domains may be sufficient to allow state-dependent conformational spread from one receptor to its neighbor (Bray and Duke, 2004).

Footnotes

  • Research in our laboratories is supported by the Medical Research Council (MRC), the European Molecular Biology Organization, the Human Frontiers Science Program, and National Institutes of Health Grants NS11756 and DA17279. J.A.F. was supported by an MRC Studentship, R.N. was supported by a postdoctoral fellowship from the California Tobacco-Related Disease Research Program and the Elizabeth Ross Fund, and D.N.B. was supported in part by a European Molecular Biology Organization Fellowship. We thank Julian Revie for constructing some of the nicotinic ACh receptor cDNAs. Requests for cDNAs should be addressed to Baljit S. Khakh (bsk{at}mrc-lmb.cam.ac.uk) and Henry A. Lester (lester{at}caltech.edu).

  • Correspondence should be addressed to Baljit S. Khakh, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. E-mail: bsk{at}mrc-lmb.cam.ac.uk.

  • Copyright © 2005 Society for Neuroscience 0270-6474/05/256911-10$15.00/0

References

  1. ↵
    Aschrafi A, Sadtler S, Niculescu C, Rettinger J, Schmalzing G (2004) Trimeric architecture of homomeric P2X(2) and heteromeric P2X(1+2) receptor subtypes. J Mol Biol 342: 333-343.
    OpenUrlCrossRefPubMed
  2. ↵
    Barajas-Lopez C, Espinosa-Luna R, Zhu Y (1998) Functional interactions between nicotinic and P2X channels in short-term cultures of guinea-pig submucosal neurons. J Physiol (Lond) 513: 671-683.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Barajas-Lopez C, Montano LM, Espinosa-Luna R (2002) Inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons. Am J Physiol Gastrointest Liver Physiol 283: G1238-G1248.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Barrera NP, Ormond SJ, Henderson RM, Murrell-Lagnado RD, Edwardson JM (2005) Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize. J Biol Chem 280: 10759-10765.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Boue-Grabot E, Barajas-Lopez C, Chakfe Y, Blais D, Belanger D, Emerit MB, Seguela P (2003) Intracellular cross talk and physical interaction between two classes of neurotransmitter-gated channels. J Neurosci 23: 1246-1253.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Boue-Grabot E, Emerit MB, Toulme E, Seguela P, Garret M (2004a) Cross-talk and co-trafficking between rho1/GABA receptors and ATP-gated channels. J Biol Chem 279: 6967-6975.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Boue-Grabot E, Toulme E, Emerit MB, Garret M (2004b) Subunit specific coupling between GABA type A and P2X2 receptor channels. J Biol Chem 279: 52517-52525.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Bray D, Duke T (2004) Conformational spread: the propagation of allosteric states in large multiprotein complexes. Annu Rev Biophys Biomol Struct 33: 53-73.
    OpenUrlCrossRefPubMed
  9. ↵
    Bunt G, Wouters FS (2004) Visualization of molecular activities inside living cells with fluorescent labels. Int Rev Cytol 237: 205-277.
    OpenUrlPubMed
  10. ↵
    Chang G, Spencer RH, Lee AT, Barclay MT, Rees DC (1998) Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science 282: 2220-2226.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Chaumont S, Jiang LH, Penna A, North RA, Rassendren F (2004) Identification of a trafficking motif involved in the stabilization and polarization of P2X receptors. J Biol Chem 279: 29628-29638.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Chavez-Noriega LE, Gillespie A, Stauderman KA, Crona JH, Claeps BO, Elliot KJ, Reid RT, Rao TS, Velicelebi G, Hapold MM, Johnson EC, Corey-Naeve J (2000) Characterization of the recombinant human neuronal nicotinic acetylcholine receptors alpha3beta2 and alpha4beta2 stably expressed in HEK 293 cells. Neuropharmacology 39: 2543-2560.
    OpenUrlCrossRefPubMed
  13. ↵
    da Fonseca PC, Morris SA, Nerou EP, Taylor CW, Morris E, P (2003) Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis. Proc Natl Acad Sci USA 100: 3936-3941.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Diaz-Hernandez M, Pintor J, Castro E, Miras-Portugal MT (2002) Colocalisation of functional nicotinic and ionotropic nucleotide receptors in isolated cholinergic synaptic terminals. Neuropharmacology 42: 20-33.
    OpenUrlCrossRefPubMed
  15. ↵
    Diaz-Hernandez M, Sanchez-Nogueiro J, Pintor J, Miras-Portugal MT (2004) Interaction between dinucleotide and nicotinic receptors in individual cholinergic terminals. J Pharmacol Exp Ther 311: 954-967.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Dickinson ME, Bearman G, Tille S, Lansford R, Fraser SE (2001) Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. Biotechniques 31: 1272, 1274-1276, 1278.
    OpenUrlPubMed
  17. ↵
    Egan TM, Khakh BS (2004) Contribution of calcium ions to P2X channel responses. J Neurosci 24: 3413-3420.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Egan TM, Haines WR, Voigt MM (1998) A domain contributing to the ion channel of ATP-gated P2X2 receptors identified by the substituted cysteine accessibility method. J Neurosci 18: 2350-2359.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Engelman HS, MacDermott AB (2004) Presynaptic ionotropic receptors and control of transmitter release. Nat Rev Neurosci 5: 135-145.
    OpenUrlCrossRefPubMed
  20. ↵
    Fisher JA, Girdler G, Khakh BS (2004) Time resolved measurement of state specific P2X ion channel cytosolic gating motions. J Neurosci 24: 10475-10487.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Fonck C, Nashmi R, Deshpande P, Damaj MI, Marks MJ, Riedel A, Schwarz J, Collins AC, Labarca C, Lester HA (2003) Increased sensitivity to agonist-induced seizures, straub tail, and hippocampal theta rhythm in knock-in mice carrying hypersensitive α4 nicotinic receptors. J Neurosci 23: 2582-2590.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Fujiwara Y, Kubo Y (2004) Density-dependent changes of the pore properties of the P2X2 receptor channel. J Physiol (Lond) 558: 31-43.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Gines S, Hillion J, Torvinen M, Le Crom S, Casado V, Canela E, Rondin S, Lew JY, Watson S, Zoli M, Agnati LF, Verniera P, Lluis C, Ferre S, Fuxe K, Franco R (2000) Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. Proc Natl Acad Sci USA 97: 8606-8611.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Green T, Heinemann SF, Gusella JF (1998) Molecular neurobiology and genetics: investigation of neural function and dysfunction. Neuron 20: 427-444.
    OpenUrlCrossRefPubMed
  25. ↵
    Higgins MK, Weitz D, Warne T, Schertler GF, Kaupp UB (2002) Molecular architecture of a retinal cGMP-gated channel: the arrangement of the cytoplasmic domains. EMBO J 21: 2087-2094.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Hille B (2001) Ion channels of excitable membranes, Chap 12, Ed 3. Sunderland, MA: Sinauer Associates.
  27. ↵
    Jiang Y, Lee A, Chen J, Cadene M, Chait BT, MacKinnon R (2002) Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417: 515-522.
    OpenUrlCrossRefPubMed
  28. ↵
    Kanjhan R, Housley GD, Burton LD, Christie DL, Kippenberger A, Thorne PR, Luo L, Ryan AF (1999) Distribution of the P2X2 receptor subunit of the ATP-gated ion channels in the rat central nervous system. J Comp Neurol 407: 11-32.
    OpenUrlCrossRefPubMed
  29. ↵
    Khakh BS (2001) Molecular physiology of P2X receptors and ATP signalling at synapses. Nat Rev Neurosci 2: 165-174.
    OpenUrlCrossRefPubMed
  30. ↵
    Khakh BS, Egan TM (2005) Contribution of transmembrane regions to ATP-gated P2X2 channel permeability dynamics. J Biol Chem 280: 6118-6129.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Khakh BS, Henderson G (2000) Modulation of fast synaptic transmission by presynaptic ligand-gated cation channels. J Auton Nerv Syst 81: 110-121.
    OpenUrlCrossRefPubMed
  32. ↵
    Khakh BS, Proctor WR, Dunwiddie TV, Labarca C, Lester HA (1999) Allosteric control of gating and kinetics at P2X4 receptor channels. J Neurosci 19: 7289-7299.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Khakh BS, Zhou X, Sydes J, Galligan JJ, Lester HA (2000) State-dependent cross-inhibition between transmitter-gated cation channels. Nature 406: 405-410.
    OpenUrlCrossRefPubMed
  34. ↵
    Khakh BS, Smith WB, Chiu CS, Ju D, Davidson N, Lester HA (2001) Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors. Proc Natl Acad Sci USA 98: 5288-5293.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Kuo A, Gulbis JM, Antcliff JF, Rahman T, Lowe ED, Zimmer J, Cuthbertson J, Ashcroft FM, Ezaki T, Doyle DA (2003) Crystal structure of the potassium channel KirBac1.1 in the closed state. Science 300: 1922-1926.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Labarca C, Schwarz J, Deshpande P, Schwarz S, Nowak MW, Fonck C, Nashmi R, Kofuji P, Dang H, Shi W, Fidan M, Khakh BS, Chen Z, Bowers BJ, Boulter J, Wehner JM, Lester HA (2001) Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety. Proc Natl Acad Sci USA 98: 2786-2791.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Lansford R, Bearman G, Fraser SE (2001) Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy. J Biomed Opt 6: 311-318.
    OpenUrlCrossRefPubMed
  38. ↵
    Le KT, Babinski K, Séguéla P (1998) Central P2X4 and P2X6 channel subunits coassemble into a novel heteromeric ATP receptor. J Neurosci 18: 7152-7159.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Lee FJ, Xue S, Pei L, Vukusic B, Chery N, Wang Y, Wang YT, Niznik HB, Yu XM, Liu F (2002) Dual regulation of NMDA receptor functions by direct protein-protein interactions with the dopamine D1 receptor. Cell 111: 219-230.
    OpenUrlCrossRefPubMed
  40. ↵
    Lester HA, Dibas MI, Dahan DS, Leite JF, Dougherty DA (2004) Cys-loop receptors: new twists and turns. Trends Neurosci 27: 329-336.
    OpenUrlCrossRefPubMed
  41. ↵
    Liu F, Wan Q, Pristupa ZB, Yu X-M, Wang YT, Niznik HB (2000) Direct protein-protein coupling enables cross-talk between dopamine D5 and g-aminobutyric acid A receptors. Nature 403: 274-280.
    OpenUrlCrossRefPubMed
  42. ↵
    MacDermott AB, Role LW, Siegelbaum SA (1999) Presynaptic ionotropic receptors and the control of transmitter release. Annu Rev Neurosci 22: 443-485.
    OpenUrlCrossRefPubMed
  43. ↵
    Michalet X, Kapanidis AN, Laurence T, Pinaud F, Doose S, Pflughoefft M, Weiss S (2003) The power and prospects of fluorescence microscopies and spectroscopies. Annu Rev Biophys Biomol Struct 32: 161-182.
    OpenUrlCrossRefPubMed
  44. ↵
    Miyazawa A, Fujiyoshi Y, Stowell M, Unwin N (1999) Nicotinic acetylcholine receptor at 4.6 A resolution: transverse tunnels in the channel wall. J Mol Biol 288: 765-786.
    OpenUrlCrossRefPubMed
  45. ↵
    Nakazawa K (1994) ATP-activated current and its interaction with acetylcholine-activated current in rat sympathetic neurons. J Neurosci 14: 740-750.
    OpenUrlAbstract
  46. ↵
    Nashmi R, Dickinson ME, McKinney S, Jareb M, Labarca C, Fraser SE, Lester HA (2003) Assembly of α4β2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons. J Neurosci 23: 11554-11567.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    Nelson ME, Kuryatov A, Choi CH, Zhou Y, Lindstrom J (2003) Alternate stoichiometries of a4b2 nicotinic acetylcholine receptors. Mol Pharmacol 63: 332-341.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    North RA (1996a) Families of ion channels with two hydrophobic segments. Curr Opin Cell Biol 8: 474-483.
    OpenUrlCrossRefPubMed
  49. ↵
    North RA (1996b) P2X receptors: a third major class of ligand-gated ion channels. Ciba Found Symp 198: 91-105.
    OpenUrlPubMed
  50. ↵
    North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82: 1013-1067.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    Patterson GH, Piston DW, Barisas BG (2000) Forster distances between green fluorescent protein pairs. Anal Biochem 284: 438-440.
    OpenUrlCrossRefPubMed
  52. ↵
    Riven I, Kalmanzon E, Segev L, Reuveny E (2003) Conformational rearrangements associated with the gating of the G protein-coupled potassium channel revealed by FRET microscopy. Neuron 38: 225-235.
    OpenUrlCrossRefPubMed
  53. ↵
    Rocheville M, Lange DC, Kumar U, Patel SC, Patel RC, Patel YC (2000) Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity. Science 288: 154-157.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Rogers M, Colquhoun LM, Patrick JW, Dani JA (1997) Calcium flux through predominantly independent purinergic ATP and nicotinic acetylcholine receptors. J Neurophysiol 77: 1407-1417.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Searl TJ, Silinsky EM (1998) Cross-talk between apparently independent receptors. J Physiol (Lond) 513: 629-630.
    OpenUrlFREE Full Text
  56. ↵
    Searl TJ, Redman RS, Silinsky EM (1998) Mutual occlusion of P2X ATP receptors and nicotinic receptors on sympathetic neurons of the guinea-pig. J Physiol (Lond) 510: 783-791.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    Shimoda K, Sauve Y, Marini A, Schwartz JP, Commissiong JW (1992) A high percentage yield of tyrosine hydroxylase-positive cells from rat E14 mesencephalic cell culture. Brain Res 586: 319-331.
    OpenUrlCrossRefPubMed
  58. ↵
    Sokolova O, Kolmakova-Partensky L, Grigorieff N (2001a) Three-dimensional structure of a voltage-gated potassium channel at 2.5 nm resolution. Structure (Camb) 9: 215-220.
  59. ↵
    Sokolova E, Nistri A, Giniatullin R (2001b) Negative cross talk between anionic GABAA and cationic P2X ionotropic receptors of rat dorsal root ganglion neurons. J Neurosci 21: 4958-4968.
    OpenUrlAbstract/FREE Full Text
  60. ↵
    Steyer JA, Almers W (2001) A real-time view of life within 100 nm of the plasma membrane. Nat Rev Mol Cell Biol 2: 268-275.
    OpenUrlCrossRefPubMed
  61. ↵
    Torres G, Egan T, Voigt M (1999a) Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners. J Biol Chem 274: 6653-6659.
    OpenUrlAbstract/FREE Full Text
  62. ↵
    Torres GE, Egan TM, Voigt MM (1999b) Identification of a domain involved in ATP-gated ionotropic receptor subunit assembly. J Biol Chem 274: 22359-22365.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67: 509-544.
    OpenUrlCrossRefPubMed
  64. ↵
    Zhou X, Galligan JJ (1998) Non-additive interaction between nicotinic cholinergic and P2X purine receptors in guinea-pig enteric neurons in culture. J Physiol (Lond) 513: 685-697.
    OpenUrlAbstract/FREE Full Text
  65. ↵
    Zwart R, Vijverberg HP (1998) Four pharmacologically distinct subtypes of a4b2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes. Mol Pharmacol 54: 1124-1131.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Email
Print
View Full Page PDF
Citation Tools
Respond to this article
An Angstrom Scale Interaction between Plasma Membrane ATP-Gated P2X2 and α4β2 Nicotinic Channels Measured with Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Microscopy
Baljit S. Khakh, James A. Fisher, Raad Nashmi, David N. Bowser, Henry A. Lester
Journal of Neuroscience 20 July 2005, 25 (29) 6911-6920; DOI: 10.1523/JNEUROSCI.0561-05.2005
del.icio.us logo Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo

Jump to section

Responses to this article

Respond to this article

Jump to comment:

No eLetters have been published for this article.