The goal of this study was to test whether any of the negatively charged residues in the extracellular domain of P2X2 might directly contribute to zinc binding. As an initial test of this idea, we made alanine mutations at all 34 extracellular positions at which either a glutamate or an aspartate was present, characterized the concentration response relation to ATP of each mutant and then carried out assays for zinc potentiation and zinc inhibition.
General effects of mutation of negatively charged residues of P2X2
When the sequences of the seven rat P2X receptors are compared, 7 of the 34 positions we tested have negatively charged residues only in P2X2. Of the remaining positions tested, sixteen have a negatively charged residue in two to four subunits, three have a negatively charged residue in five or six subunits, and eight have a negatively charged residue in all seven subunits (Table 1). When alanine was substituted at each of the 23 negatively charged positions that are not highly conserved, there were only modest changes in the concentration response relations for ATP. Similarly, although conservation between multiple members of a gene family often indicates that a residue plays an essential role, we found only modest changes in the concentration response relations for ATP when the 11 most highly conserved residues were replaced with alanines. This agrees with previous work on all of the homologous residues in human P2X1 (Ennion et al. 2001) and many of the same residues in rat P2X2 (Jiang et al. 2000). Similar to the previous work, the mutants with the most distinctive phenotypes in our study were E85A and D315A (which are equivalent to D89A and D316A in human P2X1). In oocytes, both the P2X1 D89A and the P2X2 E85A mutants produce a modest rightward shift in ATP potency. A previous attempt to express P2X2 E85A and D261A in HEK293 cells resulted in no detectable current (Jiang et al. 2000). Perhaps, the ability to express very high levels of protein in oocytes accounted for our ability to observe currents from these mutants. In all three studies, the most left-shifted mutant of the 11 conserved extracellular acidic residues was produced by placing an alanine at the position equivalent to D315 of rat P2X2. All changes at this position are not equivalent, as mutation to valine by Nakazawa et al. (1998) resulted in a 60-fold decrease in ATP potency. In summary, neither the conserved nor the unconserved negative residues of the extracellular domain could be demonstrated to play an essential direct role in allowing P2X2 receptors to respond to ATP. This stands in stark contrast to the dramatic effect on the EC50 caused by mutating some of the conserved positive residues of P2X receptors (Ennion et al. 2000; Jiang et al. 2000).
Are any negatively charged resides involved in binding zinc?
If a residue is required for binding zinc to the potentiating site, our expectation was that when the residue was replaced with an alanine the enhancement of ATP-evoked currents by 20 μM zinc would be substantially attenuated. Of the 34 positions tested in our alanine scan, seven (D82, E85, E91, E115, D136, D209, and D281) showed substantial attenuation of zinc potentiation, although all showed normal pH potentiation. Similarly, if a residue was required for binding to the inhibitory site, then the attenuation of ATP-evoked currents in the presence of 1000 μM zinc would be significantly lessened. Only one mutant, E84A, passed this preliminary test. Each of these eight candidate residues was subjected to a secondary screen which involved producing mutants that carried a cysteine at each site under study and then testing the effect of MTSET on zinc modulation. The rationale for this approach was that if the candidate residue is part of the zinc-binding site, modification with a bulky compound like MTSET should occlude the site, and weaken zinc binding.
The candidate for participation in the inhibitory zinc-binding site, E84, can be ruled out as a participant in zinc-binding, because MTSET treatment of oocytes expressing E84C resulted in greater zinc inhibition. If position 84 contributed directly to zinc-binding at the inhibitory site, less zinc inhibition would be the expected result. It was also noted that zinc inhibition was significantly enhanced when alanine was placed in eight positions. Because the zinc concentration response relations for potentiation and inhibition overlap (Clyne et al. 2002a), it was not a surprise that some mutations that depressed zinc potentiation (E85A, E91A, E115A, and D136A) showed enhanced inhibition, as no change to the inhibitory process would be needed to obtain this result. It is unclear how mutations D127A, E167A, D172A and D261A enhanced inhibition while producing no significant reduction of potentiation. It was not surprising that the E84A mutation, which had a decreased ability to produce inhibition to high zinc did not result in enhanced potentiation to 20 μM zinc, because very little inhibition is produced in wild-type oocytes by 20 μM zinc (Clyne et al. 2002a).
Of the seven candidates for participation in the potentiating site, only D136C showed significant attenuation of zinc potentiation following MTSET treatment. However, even when the MTSET effect had reached saturation (as determined by a failure to change when the duration of MTSET exposure was doubled), substantial zinc potentiation remained after MTSET treatment. Like the modification of D136C we report here, MTSET modification of H120C and H213C, residues known to be in the zinc-binding site, produces only attenuation and not elimination of zinc potentiation. One potential explanation is that although P2X2 receptors are homotrimers with three zinc-binding sites producing potentiation, it may not be possible to simultaneously modify all three zinc-binding sites with MTSET. If this is so, residual zinc potentiation would be expected after MTSET treatment, because it was previously demonstrated that even a single zinc-binding site is sufficient to produce substantial potentiation (Nagaya et al. 2005).
Of the other six candidates for involvement in zinc binding to the excitatory site, the cysteine mutant E115C did not potentiate to zinc. Additional experiments revealed that it is unlikely that E115 is in the potentiating zinc-binding site, as MTSES treatment restored the ability to respond to zinc, while attaching such a large molecule to a cysteine in the zinc-binding site would have been expected to occlude zinc binding. There are two possible explanations why the cysteine mutants of the other five candidates did not have altered responses to zinc following MTSET treatment. A simple one is that MTSET bound to the cysteine, but had no effect because the candidate residue was not near the zinc-binding site. An alternative is that one or more of these residues are part of the excitatory zinc-binding site, but MTSET was not able to access the cysteine replacing the endogenous residue. This second explanation seems unlikely because we know that MTSET can access cysteines replacing the two histidines (H120 or H213) that are known to be part of the zinc-binding site (Nagaya et al. 2005). If these six candidates are not direct participants in zinc binding, what else might have caused alanine mutations at these sites to attenuate zinc potentiation? A plausible idea is that these negative charges are essential for the normal execution of the conformational changes that follow zinc binding, but not for opening the channel in response to ATP alone, or to potentiation by acidic pH, both of which are relatively normal in these mutants.
If D136 proves to be part of the excitatory zinc-binding site, then one additional residue likely remains to be discovered, as most zinc sites are tetrahedral, and two histidines had previously been identified as participating in zinc binding. It is potentially of interest that the position equivalent to D136 is also negatively charged in P2X1, P2X4, and P2X7. P2X4 is able to potentiate in response to zinc (Wildman et al. 1999a; Xiong et al. 1999), while P2X1 and P2X7 are inhibited by zinc (Virginio et al. 1997; Wildman et al. 1999b). None of these subunits contain histidines at positions equivalent to H120 or H213 of P2X2, so if the aspartate equivalent to D136 plays a role in zinc potentiation in P2X4, one would have to look elsewhere in the sequence of this subunit for the rest of the excitatory binding site. Similarly, if E84 proves to be involved in binding zinc at the inhibitory site of P2X2, one would have to seek a different site to explain zinc inhibition of P2X1 and P2X7, as a negative residue is not found at the equivalent position in these subunits (nor in any subunit other than P2X2).