The discovery of (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile, a potent and selective agonist of human transient receptor potential cation channel subfamily m member 5 (TRPM5) and evaluation of as a potential gastrointestinal prokinetic agent.

This publication details the discovery of a series of selective transient receptor potential cation channel subfamily M member 5 (TRPM5) agonists culminating with the identification of the lead compound (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile (39). We describe herein our biological rationale for agonism of the target, the examination of the then current literature tool molecules, and finally the process of our discovery starting with a high throughput screening hit through lead development. We also detail the selectivity of the lead compound 39 versus related family members TRPA1, TRPV1, TRPV4, TRPM4 and TRPM8, the drug metabolism and pharmacokinetics (DMPK) profile and in vivo efficacy in a mouse model of gastrointestinal motility.

Cytomegalovirus (CMV) genotype in allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Based on sequence variation in the UL55 gene that encodes glycoprotein B (gB), human cytomegalovirus (CMV) can be classified into four gB genotypes. Previous studies have suggested an association between CMV gB genotype and clinical outcome in patients who underwent an allogeneic hematopoietic stem cell transplant (HSCT). The goals of this study were identify patients with active infection caused by CMV in recipients of HSCT; determine the prevalence of CMV genotypes in the study group; correlate genotype with CMV disease, acute GVHD and overall survival. METHODS: The diagnosis of active CMV infection after allogeneic HSCT was detected by antigenemia (AGM) and/or nested-PCR (N-PCR). Positive samples from patients with active CMV infection were submitted to genotyping using N-PCR to amplify a region of UL55, followed by restriction analysis based on HinfI and RsaI digestion. Real-time PCR (qPCR) was used to determine the viral load during active CMV infection and antiviral treatment. RESULTS: Sixty-three allogeneic HSCT recipients were prospectively evaluated; 49/63 (78%) patients were infected with CMV genotypes - gB1 19/49 (39%), gB2 17/49 (35%), gB3 3/49 (6%), gB4 7/49 (14%) - and 3 (6%) had mixed CMV genotypes (gB1 + gB3, gB1 + gB4 and gB2 + gB4). Characterized by gastrointestinal disease, CMV disease occurred in 3/49 (6.1%) patients, who had CMV gB3 genotype. These gB3 genotype patients presented an increasing AGM number, mean 125 (± 250) (P = 0.70), and qPCR copies/ml, mean 37938 (SD ± 50542) (P = 0.03), during antiviral treatment, when compared with other CMV genotypes. According to CMV genotypes, stratified overall survival was 55% for gB1, 43% for gB2; 0% for gB3 and 57% for gB4 (P = 0.03). CONCLUSIONS: One of the restrictions of the presented study was the low number of CMV gB sub-cohorts). However, we demonstrated that the frequency of active CMV infection in this HSCT population was high, and the most prevalent genotype in these patients with active CMV infection was gB1 and gB2 genotype (74%). In Brazil, HSCT recipients seem to carry mainly gB1 and gB2 CMV genotype.

Pore dilation of neuronal P2X receptor channels.

P2X receptors are ligand-gated ion channels activated by the binding of extracellular adenosine 5'-triphosphate (ATP). Brief (< 1 s) applications of ATP to nodose ganglion neurons or to cells transfected with P2X2 or P2X4 receptor cDNAs induce the opening of a channel selectively permeable to small cations within milliseconds. We now show that, during longer ATP application (10-60 s), the channel also becomes permeable to much larger cations such as N-methyl-D-glucamine and the propidium analog YO-PRO-1. This effect is enhanced in P2X2 receptors carrying point mutations in the second transmembrane segment. Progressive dilation of the ion-conducting pathway during prolonged activation reveals a mechanism by which ionotropic receptors may alter neuronal function.

Voltage-dependent cationic channels formed by a cytolytic toxin produced by Gardnerella vaginalis.

A cytolytic toxin produced by G. vaginalis was incorporated in artificial membranes and giant liposomes. The toxin formed ionic channels when incorporated in lipid bilayers. The electrical properties of such channels were studied. Current records revealed a unitary conductance of 126 pS (in symmetrical 150 mM KCl). The open state probability of the cytolysin formed channels was a function of the applied membrane potential. The permeability ratio of cations to anions was estimated to be 6.5.

Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor.

The P2X7 receptor is a uniquely bifunctional molecule through which ATP can open a small cationic channel typical of ionotropic receptors and also induce a large pore permeable to high molecular weight molecules (> 600 Da). Activation of this large pore can lead to cell lysis within 1-2 min. We asked whether pharmacological differences existed between the cationic channel and the cell permeabilizing pore by measuring whole-cell currents and uptake of a propidium dye (YO-PRO; Mw 629) in HEK293 cells stably expressing the rat P2X7 receptor, and comparing the actions of divalent cations and protons in these two assays. Currents in response to 2'-3'-(O)-(4-benzoyl benzoyl) ATP (BzATP, 30 microM) were inhibited by extracellular calcium, magnesium, zinc, copper and protons with half-maximal inhibitory concentrations (IC50) of 2.9 mM, 0.5 mM, 11 microM, 0.5 microM and 0.4 microM, respectively. The inhibition was voltage independent in each case. YO-PRO uptake induced by BzATP was also inhibited with similar IC50 values. The rank order of potency of a range of divalents was Cu2+ > Cd2+ = Zn2+ > Ni2+ >> Mg2+ = Co2+ > Mn2+ > Ca2+ = Ba2+ >> Sr2+. These results suggest that these divalent cations and protons all act primarily as allosteric modulators to alter the affinity of ATP binding to the P2X7 receptor. In contrast, extracellular (but not intracellular) calmidazolium inhibited the BzATP-evoked current by up to 90% (IC50 = 15 nM) but had no effect on YO-PRO uptake. Thus, calmidazolium can block activation of the ionic channel but this does not prevent the formation of the large permeabilizing pore.

Single-channel properties of cloned cGMP-activated channels from retinal rods.

Single-channel properties of a cloned channel activated by cyclic GMP have been analysed. The mRNA encoding for the channel was injected into oocytes of Xenopus laevis and the current flowing through a single ionic channel activated by cGMP was studied in excised patches under voltage-clamp conditions. The ionic channel activated by cGMP had a single-channel conductance of 32 +/- 2 pS at +120 mV and 25 +/- 4 pS at -120 mV, and its conductance was not significantly affected by increasing the cGMP concentration from 20 microM to 200 microM. The single-channel currents in the presence of NH+4, Na+, K+, Li+ and Rb+ in the medium bathing the cytoplasmic side of the membrane at +140 mV were 5.3, 4.7, 3.8, 1.3 and 0.8 pA, respectively. The single-channel current in the presence of Cs+ was less than 0.5 pA. Ca2+ and Mg2+ (both 0.5 mM) in the presence of 100 microM cGMP did not appreciably affect the channel activity at membrane potentials more negative than -80 mV, whereas at +100 mV they reduced the single-channel conductance by about threefold. The ionic selectivity and the blockage by divalent cations of the native channel found in amphibian rods and in the cloned channel from bovine rods are quite similar. However, the cloned channel has well-resolved openings, especially at positive membrane voltages, whereas the native channel is characterized by a continuous flickering between the open and closed state.

Spontaneous GABA-mediated synaptic currents in cerebellar granule cells in culture.

The whole-cell configuration of the patch clamp technique was used to characterize the electrophysiological properties of spontaneous GABA-mediated synaptic currents in cerebellar granule cells grown in a low (5 mM) potassium medium. In the presence of kynurenic acid (1 mM), to block the excitatory drive, bicuculline-sensitive synaptic events were recorded. Their amplitude distribution could be fitted by several Gaussians having the same interpeak distance. In tetrodotoxin (TTX, 1 microM) spontaneous miniature events occurred at a lower frequency. Spontaneous currents reversed polarity at 8.17 +/- 0.63 mV, a potential close to ECl; the decay phase could be fitted with a single exponential having at -60 mV a time constant of 48.7 +/- 1.5 ms. In low noise recordings, channel closing could be resolved during the decay phase of miniature events. It appeared that a single quantum of GABA opened few channels on the postsynaptic membrane.

Glycine-activated whole cell and single channel currents in rat cerebellar granule cells in culture.

The patch clamp technique was used to study whole cell and single channel currents evoked by glycine in cerebellar granule cells in culture. Whole cell concentration response curve gave a Kd value for glycine of 73 microM and a Hill slope of 1.58. Glycine-activated currents reversed close to the predicted Cl- equilibrium potential. The responses to glycine were antagonized by strychnine and picrotoxin with an IC50 of 58 nM and 172 microM, respectively. Furthermore, glycine-evoked currents were potentiated by zinc in a dose-dependent way. In outside-out membrane patches, glycine opened channels with conductances of 32, 52, 84 and 96 pS. The most frequently occurring was the 52 pS channel. The single channel current/voltage relationship was linear in the potential range between -60 and 60 mV. The 52, 84 and 96 pS channels exhibited prolonged openings whereas the 32 pS was characterized by fast (< 10 ms) openings. Open and closed time histograms of the 52 pS channel could be fitted with the sum of two or three exponentials, respectively, whereas burst duration histograms could be fitted with the sum of two exponentials. Glycine current density change drastically during days in culture, the maximal expression being between day 4 and 7, suggesting that the expression of glycine receptor channels is developmentally regulated.

Functional expression of voltage dependent sodium channels in Xenopus oocytes injected with mRNA from neonatal or adult rat brain.

The two electrode voltage clamp technique was used to study voltage-dependent sodium currents (INa) in Xenopus laevis oocytes previously injected with mRNA extracted from adult (A) or neonatal (N, < 5 days old) rat brains. In the presence of niflumic acid (300 microM) to block endogenous Ca(2+)-activated Cl- currents, depolarizing voltage steps from a holding potential of -100 mV to various voltages elicited in both groups of oocytes fast inward sodium currents which peaked at approximately 0 mV and then slowly declined to approximately 75% of the maximum current at +40 mV. At the peak, A INa was significantly larger than N INa (296 +/- 59 nA vs. 147 +/- 32 nA). Inactivation kinetics of N INa was best fit with one exponential component whereas A INa with two exponential components. A significant difference in the voltage dependence of inactivation was found between A INa or N INa. The values of Vh were -53 +/- 0.9 mV or -59.8 +/- 0.7 mV for A INa or N INa respectively. The recovery from inactivation was fitted in both groups with two exponential functions (tau f and tau s) whose values were not significantly different. However the ratio between tau f and tau s was significantly higher for N INa comparing to A INa (5.7 vs. 2.1). TTX reversibly blocked INa. The IC50 value was 58.2 +/- 6.3 nM for A INa and 20.4 +/- 2.2 nM for N INa. These results suggest that different isoforms of TTX-sensitive, voltage-dependent sodium channel subunits are functionally expressed, may be in different proportions in oocytes injected with A or N mRNA.

Characterization of three diaminopyrimidines as potent and selective antagonists of P2X3 and P2X2/3 receptors with in vivo efficacy in a pain model.

BACKGROUND AND PURPOSE: P2X3 and P2X2/3 receptors are highly localized on the peripheral and central pathways of nociceptive signal transmission. The discovery of A-317491 allowed their validation as chronic inflammatory and neuropathic pain targets, but this molecule has a very limited oral bioavailability and CNS penetration. Recently, potent P2X3 and P2X2/3 blockers with a diaminopyrimidine core group and better bioavailability were synthesized and represent a new opportunity for the validation of P2X3-containing receptors as targets for pain. Here we present a characterization of three representative diaminopyrimidines. EXPERIMENTAL APPROACH: The activity of compounds was evaluated in intracellular calcium flux and electrophysiological recordings from P2X receptors expressed in mammalian cells and in a in vivo model of inflammatory pain (complete Freund's adjuvant (CFA) in rat paws). KEY RESULTS: Compound A potently blocked P2X3 (pIC(50)= 7.39) and P2X2/3 (pIC(50)=6.68) and showed no detectable activity at P2X1, P2X2, P2X4 and P2X7 receptors (pIC(50)< 4.7). Whole-cell voltage clamp electrophysiology confirmed these results. Compounds showed good selectivities when tested against a panel of different classes of target. In the CFA model, compound B showed significant anti-nociceptive effects (57% reversal at 3mg·kg(-1) ). CONCLUSIONS AND IMPLICATIONS: The diaminopyrimidines were potent and selective P2X3 and P2X2/3 receptor antagonists, showing efficacy in vivo and represent useful tools to validate these receptors as targets for inflammatory and neuropathic pain and provide promising progress in the identification of therapeutic tools for the treatment of pain-related disorders.

Phenotypic characterization of three clinical isolates of Burkholderia pseudomallei in Ceará, Brazil.

Burkholderia pseudomallei, the causative agent of melioidosis was found in a small cluster of cases in Tejuçuoca, Ceará, Brazil. Tests were carried out to determine its phenotypic characteristics: colony morphology on Ashdown agar and MacConkey agar, biochemical profile in conventional biochemical tests and API 20NE, arabinose assimilation and susceptibility testing by disk diffusion, comparing with data in the literature. This study confirms the presence of B. pseudomallei in Brazil and describes its characteristics.

Functionally distinct chloride-mediated GABA responses in rat cerebellar granule cells cultured in a low-potassium medium.

The patch-clamp technique was used to study whole cell currents evoked by gamma-aminobutyric acid (GABA) in rat cerebellar granule cells cultured in 5 mM potassium, a condition that favors the development of functional GABAergic synapses. GABA activated both high- and low-sensitivity receptors. The high-sensitivity receptor had an effective concentration producing half the maximum response (EC50) of 13 microM, whereas the low-sensitivity one had an EC50 of 255 microM. The GABAA receptor agonist isoguvacine activated only the high-sensitivity receptor with an EC50 of 16 microM. When GABA was applied during the desensitized phase of the response elicited by a saturating concentration of isoguvacine, it was still able to induce a small response, whereas when isoguvacine was applied during the desensitizing phase of GABA-evoked current no response was detected. GABA responses were highly heterogeneous regarding their sensitivity to bicuculline. In a small number of cells (3 of 25), bicuculline (10 microM) completely abolished GABA-evoked currents. In the majority of the neurons (22 of 25) the blocking effect of bicuculline (100 microM) was 64 +/- 4% (mean +/- SE). The bicuculline-resistant component was abolished by picrotoxin (100 microM). In bicuculline, the dose-response curve for GABA was fitted with a sigmoidal curve with an EC50 value of 209 microM. These data indicate that functional new GABA receptor types with unusual pharmacology could be switched on by conditions that maintain cells in their undifferentiated state.

Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor.

1. Extracellular ATP acting on P2X7 receptors opens a channel permeable to small cations, creates an access pathway for the entry of larger molecular weight dyes, and causes cell death. We used whole-cell recording and fluorescence microscopy to measure the time courses of ionic currents, uptake of the propidium dye YO-PRO-1, and membrane disruption, in human embryonic kidney (HEK293) cells expressing the rat P2X7 receptor. 2. The ATP analogue 2', 3'-O-(benzoyl-4-benzoyl)-ATP (30 microM) induced membrane blebbing within 30-40 s of sustained application; this was 5-10 times slower when extracellular sodium was replaced by larger cations. 3. Fluorescence of YO-PRO-1 was detectable within 3 s, and the uptake reached a steady rate within 10-20 s; YO-PRO-1 uptake was greatly enhanced by removing extracellular sodium. 4. Electrophysiological measurements of current reversal potentials with intracellular sodium and extracellular cations of different sizes showed that the ionic channel progressively t'2+LE0i%-i"dilated during 10-20 s to a diameter greater than 1 nm (10 A). With short agonist applications (3-5 s) the pore dilatation and YO-PRO-1 uptake were reversible and repeatable. 5. Polyethylene glycols having molecular weights >= 5000 blocked the increase in cation permeability, YO-PRO-1 uptake and membrane blebbing. 6. We conclude that maximum P2X7 receptor activation causes an exponential dilatation of the ion channel with a time constant of 25 s to a final diameter of 3-5 nm from an initial minimum pore diameter of 0.8 nm.

Trinitrophenyl-substituted nucleotides are potent antagonists selective for P2X1, P2X3, and heteromeric P2X2/3 receptors.

There are currently seven P2X receptor subunits (P2X1-7) defined by molecular cloning. The functional identification of these receptors has relied primarily on the potency of alpha,beta-methylene-ATP relative to that of ATP and on the kinetics of receptor desensitization. In the present experiments we found that the 2', 3'-O-(2,4,6-trinitrophenyl)-substituted analogs of ATP are selective and potent antagonists at some but not all P2X receptors. The trinitrophenyl analogs of ATP, ADP, AMP, and GTP produced a reversible inhibition of ATP-evoked currents in human embryonic kidney 293 cells expressing P2X1 receptors, P2X3 receptors, or both P2X2 and P2X3 (heteromeric) receptors; IC50 values were close to 1 nM. These compounds were at least 1000-fold less effective in blocking currents in cells expressing P2X2, P2X4, or P2X7 receptors (P2X5 and P2X6 not tested). GTP, 2,4,6-trinitrophenol, and the 2',3'-trinitrophenyl analog of adenosine (0.1-10 microM) had no effect. Thus, we have identified a structural motif that confers antagonist action at P2X receptors that contain P2X1 or P2X3 subunits (the alpha,beta-methylene-ATP-sensitive subclass).

Calcium permeability and block at homomeric and heteromeric P2X2 and P2X3 receptors, and P2X receptors in rat nodose neurones.

1. Whole-cell recordings were made from HEK 293 (human embryonic kidney) cells stably transfected with cDNAs encoding P2X2, P2X3 or both receptors (P2X2/3) and from cultured rat nodose neurones. Nodose neurones all showed immunoreactivity for both P2X2 and P2X3, but not P2X1, receptors. 2. Reversal potentials were measured in extracellular sodium, N-methyl-D-glucamine (NMDG) and NMDG containing 5 mM Ca2+; the values were used to compute relative permeabilities (PNMDG/PNa and PCa/PNa). PNMDG/PNa was not different for P2X2, P2X2/3 and nodose neurones (0.03) but was significantly higher (0.07) for P2X3 receptors. PCa/PNa was not different among P2X3, P2X2/3 and nodose neurones (1.2-1.5) but was significantly higher (2.5) for P2X2 receptors. 3. External Ca2+ inhibited purinoceptor currents with half-maximal concentrations of 5 mM at the P2X2 receptor, 89 mM at the P2X3 receptor and 15 mM at both the P2X2/3 heteromeric receptor and nodose neurones. In each case, the inhibition was voltage independent and was overcome by increasing concentrations of agonist. 4. These results may indicate that Ca2+ permeability of the heteromeric (P2X2/3) channel is dominated by that of the P2X3 subunit, while Ca2+ block of the receptor involves both P2X2 and P2X3 subunits. The correspondence in properties between P2X2/3 receptors and nodose ganglion neurones further supports the conclusion that the native alpha,beta-methylene ATP-sensitive receptor is a P2X2/3 heteromultimer.

The antagonist trinitrophenyl-ATP reveals co-existence of distinct P2X receptor channels in rat nodose neurones.

1. Whole-cell recordings were made from rat nodose ganglion neurones in culture and from human embryonic kidney (HEK293) cells stably transfected to express P2X2, P2X3 or both receptor subunits. We examined the blocking actions of 2',3'-O-trinitrophenyl-ATP (TNP-ATP) on currents evoked by the agonists ATP and alpha, beta-methylene ATP. 2. In cells expressing only P2X2 or P2X3 receptor subunits, the inhibition by TNP-ATP was fitted by a single binding site model with half-maximal concentrations of about 3 microM and 3 nM, respectively. In cells expressing both P2X2 and P2X3 receptor subunits, currents showed little or no desensitization, thus excluding contributions from homomeric P2X3 receptors. When alpha,beta-methylene ATP was the agonist (activating heteromeric P2X2/3 receptors), the inhibition by TNP-ATP conformed to a single binding site (half-maximal concentration about 3 nM). When ATP (30 microM) was the agonist, activating both heteromeric P2X2/3 as well as homomeric P2X2 receptors, the inhibition curve was biphasic (half-maximal concentrations about 3 nM and 3 microM); the proportion of high affinity sites in all six cells tested was about 40 %. 3. In nodose ganglion neurones, the inhibition by TNP-ATP of currents evoked by ATP (30 microM) was also clearly biphasic. In this case, individual neurones showed more variability in the proportion of high and low affinity sites for TNP-ATP. 4. We conclude that more than one form of multimeric P2X receptor channels are functionally expressed on the cell bodies of individual nodose ganglion neurones. On the basis of sensitivity to TNP-ATP, and other properties, one of these may correspond to the homomeric P2X2 receptor and the other(s) to heteromeric P2X2/3 receptors.

Physical characterization of the pore forming cytolysine from Gardnerella vaginalis.

The cytolytic toxin (CTox) produced by Gardnerella vaginalis is able to form voltage-dependent cationic channels when incorporated in lipid membranes (Moran et al. (1991) FEBS Lett. 283, 317-320). Osmotic protection experiments show that toxin incorporated in human erythrocytes forms pores between 18 A and 28 A in diameter. A hypothesis of pore formation as a primary event to produce cytolysis is proposed. The CTox activity increases when cells are depolarized by increasing the extracellular K+ concentration, probably reflecting the voltage dependent character of CTox formed channels. The cytolytic effect of the toxin was prevented by low temperatures and was a function of the extracellular Ca2+ concentration, suggesting a Ca2+ influx as part of the lytic mechanism. Binding of CTox to erythrocytes was dependent on external Ca2+ and was less temperature-dependent. Dose-response analysis suggests cooperativity of the toxin for the lytic activity, although no direct evidence of oligomerization has been found.

Baculovirus expression provides direct evidence for heteromeric assembly of P2X2 and P2X3 receptors.

P2X2 and P2X3 are subunits of P2X receptors, cation channels opened by binding extracellular ATP. cDNAs encoding P2X2 and P2X3 receptor subunits, each with one of two C-terminal epitope tags, were cloned into baculovirus. Virally infected insect cells (Spodoptera frugiperda) expressed moderate to high levels of the corresponding proteins, as detected by Western blotting, by the specific binding of [35S]ATP and by whole-cell recordings of membrane current evoked by ATP or alphabetamethylene-ATP. In cells infected at the same time with two viruses encoding P2X2 and P2X3 receptors, the two proteins could be cross-immunoprecipitated with antibodies specific for either of the epitope tags. Whole-cell recordings from these cells showed that ATP and alphabetamethylene-ATP evoked currents with agonist sensitivity and desensitization quite distinct from those observed when P2X2 or P2X3 receptors were expressed alone. The results offer a method to express large amounts of P2X receptor protein, and they provide direct evidence that P2X2 and P2X3 subunits assemble to form heteromeric channels having distinct properties from those formed as homomers.

The permeabilizing ATP receptor, P2X7. Cloning and expression of a human cDNA.

A cDNA was isolated from a human monocyte library that encodes the P2X7 receptor; the predicted protein is 80% identical to the rat receptor. Whole cell recordings were made from human embryonic kidney cells transfected with the human cDNA and from human macrophages. Brief applications (1-3 s) of ATP and 2', 3'-(4-benzoyl)-benzoyl-ATP elicited cation-selective currents. When compared with the rat P2X7 receptor, these effects required higher concentrations of agonists, were more potentiated by removal of extracellular magnesium ions, and reversed more rapidly on agonist removal. Longer applications of agonists permeabilized the cells, as evidenced by uptake of the propidium dye YO-PRO1, but this was less marked than for cells expressing the rat P2X7 receptor. Expression of chimeric molecules indicated that some of the differences between the rat and human receptor could be reversed by exchanging the intracellular C-terminal domain of the proteins.

Isoquinolines as antagonists of the P2X7 nucleotide receptor: high selectivity for the human versus rat receptor homologues.

1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) and N-[1-[N-methyl-p-(5 isoquinolinesulfonyl)benzyl]-2-(4 phenylpiperazine)ethyl]-5-isoquinolinesulfonamide (KN-04) potently inhibit the human lymphocyte P2Z receptor, an ATP-gated cation channel [Br J Pharmacol 120:1483-1490 (1997)]. Although the molecular identity of the lymphocyte P2Z receptor has not been established, it shares many functional characteristics with the cloned P2X7 nucleotide receptor. We have tested whether these isoquinolines inhibit P2X receptor function in human embryonic kidney 293 cells that stably express the human or rat recombinant P2X7 receptors. ATP activation of cation currents and uptake of the organic dye ethidium were potently inhibited by KN-62 and KN-04 in human embryonic kidney cells expressing the human P2X7R but not the rat P2X7R, even though these species homologues share 80% amino acid identity. Introduction of the first 335 amino acids of the human P2X7R sequence conferred KN-62 sensitivity to the rat P2X7R; this suggests that isoquinolines interact with residues in the amino-terminal half (containing the large extracellular loop) of the human P2X7R. KN-62 and KN-04 also potently inhibited ATP-gated Ca2+ influx and ethidium uptake in several leukocyte cell lines (THP-1, BAC1.2f5, and BW5147) that natively express the human or murine P2X7R mRNA. The ability of isoquinoline sulfonamides to potently inhibit human and murine P2X7R signaling will be a useful tool for identifying P2Z/P2X7 functional responses in other cell types. The substantial differences in pharmacological sensitivity between rat and human P2X7R may also indicate structural domains important in channel/pore activation.