Pharmacological and Biophysical Properties of the Human P2X₅Receptor

Abstract

We constructed a full-length human P2X 5 purinoceptor cDNA by incorporating a sequence corresponding to exon 10, which is missing in cDNAs cloned previously from human tissues. We studied the functional properties by patch-clamp recording and fluorescence imaging after expression in human embryonic kidney 293 cells. ATP (1-100 M; half-maximal current at 4 M) elicited inward currents at Ϫ60 mV; these persisted during brief (2 s) applications but declined during longer applications. The peak current was dependent on the holding potential and showed little rectification; however, both the desensitization during the application and the decline in the current when ATP was washed out were slower at ϩ30 mV than at Ϫ60 mV. 2Ј,3Ј-O-(4-Benzoyl)-benzoyl-ATP and ␣␤-methylene-ATP mimicked the action of ATP (half-maximal concentrations 6 and 161 M, respectively). The currents were inhibited by suramin, pyridoxal-5-phosphate-6-azo-2Ј,4Ј-disulfonic acid and Brilliant Blue G, with half-maximal inhibition at 3, 0.2, and 0.5 M, respectively; 2Ј,3Ј-O-(2Ј,4Ј,6Ј-trinitrophenol)-ATP (1 M) was ineffective. Removing divalent cations did not significantly alter ATP concentration-response curves. Reversal potential measurements showed that the human P2X 5 receptor was permeable to calcium (P Ca /P Na ϭ 1.5) and N-methyl-D-glucamine (NMDG) (P NMDG /P Na ϭ 0.4); it was also permeable to chloride (P Cl /P Na ϭ 0.5) but not gluconate (P gluc /P Na ϭ 0.01) ions. The permeability to NMDG developed as quickly as the channel opened, in contrast to the P2X 7 receptor where the NMDG permeability develops over several seconds. Cells expressing human P2X 5 receptors also rapidly accumulated the propidium dye YO-PRO-1 in response to ATP.A P2X 5 subunit cDNA was first isolated from libraries of rat sympathetic ganglia (Collo et al., 1996) and heart (Garcia-Guzman et al., 1996). The RNA is expressed predominantly in heart (Garcia-Guzman et al., 1996) but there is also expression in brain, spinal cord, and adrenal gland (GarciaGuzman et al., 1996). Immunoreactivity for P2X 5 subunits has also been described in developing skeletal muscle of the rat (Meyer et al., 1999;Ryten et al., 2001); recently, the receptor has been implicated in the differentiation of satellite cells into mature multinucleated muscle fibers (Ryten et al., 2002). Mammalian skin and endocrine (Glass and Burnstock, 2001) cells also show P2X 5 immunoreactivity.When expressed in HEK293 cells, the rat P2X 5 subunit cDNAs gave rise to currents activated by ATP, but these were very small and their properties not extensively studied. The current was elicited by ATP [half-maximal concentration (EC 50 ) about 10 M] but not by ␣␤meATP, and it was readily blocked by suramin and PPADS in the 1 to 10 M concentration range (Collo et al., 1996;Garcia-Guzman et al., 1996). A mouse cDNA has also been cloned and expressed; again, the currents recorded from HEK293 cells were only a few tens of picoamperes and they were not studied in detail . On the other hand, although these two mammalian recep...

The P2X7 receptor in retinal ganglion cells: A neuronal model of pressure-induced damage and protection by a shifting purinergic balance

“…The calcium elevation was considerably higher when cells were stimulated by 10 µM BzATP than by 100 µM ATP; the relative potency of BzATP over ATP is a key characteristic of P2X 7 receptors [23,24]. The threefold increase in the response to BzATP upon removal of magnesium is similar to that described for the cloned P2X 7 receptor and rules out a contribution from P2X 5 receptors [20,25]. Pharmacologic identification in rat ganglion cells was supported by the ability of 1 µM Brilliant Blue G (BBG) to completely block the calcium elevations triggered by BzATP; the neuroprotective effects of 100 nM BBG described below, combined with the lack of a contribution from the P2X 5 receptor, support the inhibition as specific for the P2X 7 receptor.…”

“…However, the lethal effects of BzATP were also inhibited by 100 nM BBG. At this concentration, BBG inhibits only 5% of the current through the P2X 5 receptor [20] and is generally considered specific for the receptor P2X 7 receptor [22]. Further confirmation came from the lack of inhibition by 30 µM suramin [19].…”

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

“…In contrast, a recent study concluded that these properties could only be measured when Panx-1 was coexpressed (18). Second, mutant P2X 2 and P2X 4 (2, 5) and human P2X 5 receptors (33) open to the I 2 state with little or no time delay and, in many cases, in oocytes that lack native Panx-1 (21). Third, in one study, Panx-1 was not part of the P2X 7 receptor signaling complex (34), whereas in another it was (16,17).…”

ENaC, renal sodium excretion and extracellular ATP

“…For example, calcium influx through P2X receptors can lead to the activation of Ca 2+ -dependent chloride currents, which can be prevented either by substituting extracellular calcium with equimolar barium or by adding chloride channel blockers (such as DIDS or NPPB) to the bathing medium. Additionally, some P2X receptors (notably, human P2X 5 ) lose their selectivity for cations with time and become increasingly permeable to chloride [38].…”