(A) Whole cell patch clamp currents evoked by changing external pH from 7.4 to 6.9 on a DiI-labeled sensory neuron that innervated skeletal muscle. The smaller pH-evoked current (left) was before and the larger (right) was 15 seconds following washout of extracellular 50 µM ATP that had been applied for 25 seconds; the first 3 seconds of ATP application is shown between the two pH-evoked currents. Scale: 1 nA, 1 sec. Inset: fluorescence (upper) and phase micrographs showing dissociated sensory neurons, two of which are fluorescent because of retrograde transport of DiI injected into thigh muscles. Scale bar: 30 µm.
(B) Distribution of muscle afferents by cell diameter (white bars). Note that larger cells were more likely to express ASIC3-like currents (grey) and that currents in 35% of ASIC3+ cells were not sensitized by ATP (black).
(C) Mean (+/− s.e.m.) percent increase (100% = 2-fold increase) in ASIC3-like current (pH 6.9) at different ATP concentrations, each applied for 1 minute (n ≥ 5 for each concentration).
(D) Mean percent increase in ASIC3-like current evoked at the indicated pH. 50 µM ATP was applied for 1 minute at pH 7.4. ASICs were evoked by brief (2 sec) steps to the indicated pH before and 20 seconds after removal of ATP. Currents at the peak of the ASIC activation curve (ca. pH 6) were almost unchanged, indicating that ATP does not increase the total number of available ASIC molecules. (n ≥ 5 for each pH).

(A) Each trio of traces is from a single COS or CHO cell transfected with the indicated cDNAs. The ASIC3 currents, evoked by 2 second steps to pH 6.9 before ATP application and after removal, flank a trace showing the first 3 seconds of a 30 second ATP application. αβ-methylene-ATP was used on the P2X2/P2X3 co-transfection to assure only activation of the heteromer and not any P2X2 homomers. Scale bars: 1 sec; 0.5 nA. See supplemental Figure S3 for time course of sensitization in CHO cells. (B) Mean percent ASIC current increase (n = 7–83 cells, each from dishes not previously exposed to ATP) in sensory neurons (DRG) or cell lines transfected with ASIC3 or ASIC1 together with the indicated P2X receptor(s). P2X2, P2X4, and P2X5 can mediate sensitization of ASIC3; ASIC1 also responds to ATP.

(A) CHO cells transfected with the indicated combination of channels. Each trio of traces shows ASIC3 currents evoked prior to ATP, after 20 seconds in 5 µM ATP plus 150 µM suramin, and after a subsequent 20 second application of 5 µM ATP alone. Suramin blocked ASIC sensitization by P2X2 (upper traces) but not by P2X4 (middle traces). After 20 seconds in ATP plus suramin, subsequent ATP application had no further effect on P2X4 cells. Cells transfected with both P2X2 and P2X4 exhibited sensitization in suramin, like P2X4, and also after suramin, like P2X2 (lower traces).
(B) Summary data (n ≥ 5 in each condition) shows that CHO cells that co-express P2X2 and P2X4 exhibited ASIC sensitization in response to the first application (ATP plus suramin) roughly equivalently to P2X4-only cells, and to the second application (ATP only) roughly equivalently to P2X2-only cells. This is consistent with independent actions of P2X2 and P2X4.

Sensory neurons in two sections taken from an L5 dorsal root ganglion. Left: section labeled and imaged for antisera to P2X5 (A), and ASIC3 (B). Right: section labeled and imaged for antisera to P2X2 (D), and ASIC3 (E). Bottom images (C and F) superimpose the two above (P2X in red, ASIC in green). Secondary antibodies: anti-guinea pig-cy3 (for P2X), anti-rabbit cy5 (for ASIC). Counts of all labeled cells were made blindly in well stained sections from 3 rats (Supplementary Table S7). For antibody validation see supplemental Figure S7.

Each pair of acid-evoked currents (pH step from 7.4 to 6.9, 4 sec) is from a single representative sensory neuron (n ≥ 5 cells tested for each condition). Currents were evoked either before or 30 sec after a 1 minute application of the indicated compound. Purinergic agonists were applied at 50 µM for 1 minute except BzATP (100 µM). Antagonists (TNP-ATP, concentration indicated; suramin, 100 µM; PPADS, 4 µM) were applied together with ATP. If ASIC current did not increase in presence of an antagonist, ATP was reapplied without antagonist and then current increased (not shown). Scale bars: 1 sec, 0.5 nA. Insensitivity to either ADP or UTP rules out P2Y receptors; insensitivity to αβmethylene-ATP rules out P2X3, P2X2/3, and P2X1. Inhibition by suramin and PPADS (not shown) is inconsistent with P2X4 and P2X7.

(A) Photodestruction of ASIC3-YFP resulted in an increase of P2X5-CFP fluorescence. Relative CFP (closed black circles, right axis) and YFP (grey squares, left axis) intensities measured on an epifluorescence microscope were plotted as a function of time of photobleaching of YFP in CHO cells co-expressing P2X5-CFP and ASIC3-YFP. When only P2X5-CFP was expressed (open circles), there was no increase in CFP fluorescence. CFP excitation and emission wavelengths centered at 436 and 485 nm; YFP at 514 and 535 nm; photobleaching at 514 nm.
(B) The increase in CFP fluorescence was proportional to the fraction of YFP that was photodestroyed. P2X5-CFP appeared closely associated with ASIC3-YFP with a FRET efficiency of approximately 20% (black circles). FRET did not occur between P2X2-CFP and NgCAM-YFP (dark grey diamonds). Yellow CaMeleon 3.1 (YC3.1) served as a positive FRET control, exhibiting a 40% FRET signal (light grey triangles). See supplemental figure S4.

(A) ASIC currents before and after exposure to ATP when extracellular Ca²⁺ was chelated during ATP application (1 mM EGTA, no added Ca²⁺ or Mg²⁺. Bath solution was returned to 2 mM Ca²⁺ and 1 mM Mg²⁺ prior to evoking ASIC currents). Sensitization did not occur in P2X2-transfected CHO cells when ATP was applied in the absence of external Ca²⁺ (2^nd set of traces), but did in sensory neurons (DRG) and in CHO cells transfected with P2X4 or P2X5. Scale bars: 1 sec; 1 nA. For signaling pathways not implicated in sensitization see supplemental Figure S6, supplemental Table S6.
(B) Three different sensory neurons that either exhibited ATP sensitization (top and bottom cells) or did not (middle). Nothing about the waveform of ATP-evoked current (middle trace in each trio) could predict whether sensitization occurred. pH steps were from 7.4 to 6.8; ATP was 50 µM (4 sec is shown of a 30 sec application). Scale bars: 1 sec; 1 nA (ASIC currents) or 0.5 nA (P2X currents).
(C) Representative amplitudes of three kinds of P2X currents each transfected with 10 µg/ml of the indicated cDNA. As previously shown by Collo et al.(Collo et al., 1996), P2X5 makes little current. Scale bars: 1 sec; 1 nA.
(D) Plot of average percent increase of ASIC current vs. the amplitude of sustained ATP-evoked current in DRG sensory neurons and COS cells transfected with ASIC3 and either P2X2 or P2X5. ASIC sensitization by ATP occurred in native neurons and P2X5-expressing COS cells although there was little sustained ATP-evoked current. In contrast, sensitization required high P2X2 currents (>2 nA) and increased linearly with P2X amplitude above 2 nA.