Covalent modification of mutant rat P2X2 receptors with a thiol-reactive fluorophore allows channel activation by zinc or acidic pH without ATP

Abstract

Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C(5)-maleimide (AM546). Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.

Figure 1. Activation of K69C and K308C mutant receptors by MTSEA.

A: Response of an oocyte expressing K69C to ATP before (thin line) and after (thick line) treatment with MTSEA. B: Similar data for an oocyte expressing K308C. The scale bar is the same as in A. C: ATP concentration-response relations for a series of oocytes studied as in A and B. The error bars represent standard error of the mean and are smaller than the symbols for some points. (N = 5–6). D–E: Response to zinc or acid jumps in the presence of 1,000 µM ATP of oocytes expressing K69C or K308C before (thin line) and after (thick line) treatment with MTSEA. F: Quantitative characterization of a series of experiments studied as in D and E (N = 3). In this figure, and most others, the average inward current is plotted as an upward bar. The exception to this convention is noted in the legend for the relevant figure.

Figure 2. AM546 activated the K69C receptor.

A–B: Sample traces from oocytes expressing either wild type rP2X2 (WT) or K69C as they were exposed to 50 µM AM546 for 5 minutes and then washed with recording solution. The horizontal dotted lines indicate the pretreatment holding current levels. The vertical dashed lines to the right of each trace indicate the difference in holding current 2 min after washout from the holding current before exposure to the drug. For wild type the change was so small that the whiskers obscure the amplitude of the dashed line. C: AM546 treatment caused K69C expressing oocytes to become responsive to zinc and acidic pH in the absence of exogenous ATP, but had no effect on responses of wild type receptors. Note the change in holding current after AM546 treatment in K69C expressing oocytes but not wild type oocytes (vertical dashed lines to the right of the traces). D–E: Quantitative characterization of a series of oocytes studied as in C, as well as control oocytes exposed to the vehicle alone (0.5% DMSO) (N = 4–8). Asterisks indicate a statistically significant difference from the DMSO control tested on the same construct.

Figure 3. Concentration-response relation of activation of K69C receptors by AM546.

A–B: Concentration-response plots of the current activated by 100 µM zinc, and current activated by pH 6.5 after incubation in AM546. The data in the plots were fit with a single-exponential rise to a maximum (N = 5–10).

Figure 4. Concentration-response relations for the effects of zinc and pH.

A: Effect of increasing zinc concentration on the currents of an oocyte expressing wild type rP2X2 in the continuous presence of 5 µM ATP. There was already a substantial inward current at 5 µM ATP with 5 µM zinc, but for quantifying the magnitude of the effect for a series of similar oocytes, the current with 5 µM zinc was defined as 0, and changes from this level are shown in the trace and the accompanying graph in C. B: Effect of increasing zinc concentration, in the absence of exogenous ATP, on the currents of an oocyte expressing K69C after treatment with AM546. The current with 5 µM zinc was defined as 0. C: Quantification of a series of oocytes studied as in A and B. The data points for wild type rP2X2 (filled circles) are scaled by the left y-axis and the data points for K69C (open circles) are scaled by the right y-axis (N = 5). D: Effect of increasing proton concentration on the currents of an oocyte expressing wild type rP2X2 in the continuous presence of 5 µM ATP. The currents at pH 8.5 were defined as 0. E: Effect of increasing proton concentration, in the absence of exogenous ATP, on the currents of an oocyte expressing K69C after treatment with AM546. The currents at pH 8.5 were defined as 0. F: Quantification of a series of oocytes studied as in D and E. The data points for wild type rP2X2 (filled circles) are scaled by the left y-axis and the data points for K69C (open circles) are scaled by the right y-axis (wild type, N = 7; K69C, N = 3).

Figure 5. Effects of voltage on wild type rP2X2 and on AM546-treated K69C receptors.

A: Representative traces of a rP2X2 expressing oocyte to a 2 s ramp from −100 to +100 mV in the presence of either 10 or 1,000 µM ATP (thin and thick traces, respectively) at pH 7.5 or pH 6.0. ATP was applied beginning approximately 5 seconds before the portion of the traces that are illustrated B: Current-voltage plots of rP2X2 receptor responses based on multiple experiments as in A. Data from ramps in the absence of ATP were subtracted from data in the presence of ATP. (N = 7). C: Representative traces of a K69C expressing oocyte to a voltage ramp at either pH 7.5 or pH 6.0 recording solution, before (thin traces) and after (thick traces) AM546 treatment. D: Current-voltage plots based on multiple experiments as in C. Data from ramps before AM546 treatment were subtracted from data taken after AM546 treatment. (N = 6).

Figure 6. Zinc and acidic pH activate AM546-treated K69C receptors to a greater degree than ATP alone.

A: Response of a K69C expressing oocyte to 1,000 µM ATP before and after AM546 (50 µM, 5 min). Traces were not baseline-corrected to emphasize the drop in holding current (vertical dashed line) after AM546 exposure. B: Effect of MTSEA challenge after treatment with AM546. The vertical dashed line indicates the drop in holding current after AM546. The horizontal dashed lines indicate the amplitude of the ATP response after MTSEA. C: Summary of a series of experiments as in B. The data are plotted as the responses after MTSEA normalized to the responses after AM546. The horizontal dashed line indicates 100% (i.e. no change in response after MTSEA). (N = 8). D: Effect of AM546 dosage (concentration * duration of exposure) on inward currents evoked by 1,000 µM ATP.

Figure 7. Effects of AM546 on the K69C/H319K double mutant.

A: Responses to acidic pH and zinc before and after AM546 treatment. The dashed lines indicate the 0 current level, and indicate that there was a large inward holding current prior to any treatment. B: Average responses for a series of cells studied as in A. (N = 16 for holding current, 8 for zinc and 4 for pH 6.5). In contrast to the other figures, inward currents are plotted as downward deflections, to emphasize the different signs of the responses to zinc and acidic pH in this mutant. C: ATP concentration-response relations before and after AM546 treatment (N = 4).

Figure 8. Lack of effect of AM546 on K308C.

A: Responses of an oocyte before and after AM546 (50 µM, 15 minutes). The cell was then treated with MTSEA to test whether the cysteines had been modified by the AM546 treatment. B: Average ATP responses for a series of cells studied as in A or control cells that received MTSEA only. There was no response to zinc or pH 6.5 before or after AM546 or MTSEA treatment.

Figure 9. Responses of AM546-treated K69C receptors to suramin.

A: Effect of suramin on an oocyte expressing wild type rP2X2 when applied without exogenous ATP. The horizontal dashed line indicates the 0 current level. B: Correlation between the initial holding current of oocytes expressing wild type rP2X2 and the amplitude of the outward current in response to suramin (20 µM) or apyrase (1 mg/ml) C: Average amplitude of the outward current in response to suramin, apyrase, or recording solution alone applied from a separate barrel of the solution switcher (control). D: Effect of suramin on an oocyte expressing K69C before AM546 (thin gray trace) and after AM546 (thick black trace). The two traces were not baseline corrected to indicate the drop in holding current caused by AM546 (vertical dashed line). E: Concentration-response plots of suramin on wild type rP2X2 and AM546-treated K69C receptors (N = 6). F: Response of a K69C oocyte to 200 µM suramin after treatment with AM546 and then retested after exposure to MTSES. G: Results of a series of experiments as in F. The “AM546 after MTSES” group represents a control to verify that the dose of MTSES given to the “MTSES after AM546” experimental group was sufficient to occupy all free cysteines.

Figure 10. Structural features likely contributing to activation of AM546 modified K69C channels by zinc or pH.

A: Structure of ATP folded as it sits in the binding site of zebrafish P2X4.1, and structure of AM546 with the linker folded back upon itself. B: Structure of P2X4.1 in the closed and open (ATP-bound) states. Only two of the three subunits are illustrated. Residues that bind ATP are shown in ball and stick format. The gray boxes superimposed on the closed state indicate domains of the receptor named according to the dolphin model of Kawate and Gouaux . Left: The closed state structure of zP2X4.1 (PDB 4DW0) with positions homologous to key residues of rP2X2 colored. Orange indicates the location of the histidines of the potentiating zinc binding site of rP2X2, which are P125 (cyan subunit) and H219 (pink subunit) in zP2X4.1. Beige is the location of F327 (of the cyan subunit), which is homologous to rP2X2 H319. Blue (L64) and purple (P199) indicate the residues (of the pink subunit) that bind to F327 in the closed state. Right: The ATP bound open state structure of zP2X4.1 (PDB 4DW1). Residues are colored as in A. The ATP at the interface between the two illustrated subunits is also shown (green). Note that the side group of H219 has been rotated from its position in 4DW1 to emphasize the close apposition of the orange histidines that is possible in rP2X2 when zinc is present.