Striated myogenesis and peristalsis in the fetal murine esophagus occur without cell migration or interstitial cells of Cajal.

Esophageal striated myogenesis progresses differently from appendicular myogenesis, but the mechanism underlying this process is incompletely understood. Early theories of transdifferentiation of smooth muscle into striated muscle are not supported by transgenic fate-mapping experiments; however, the origin of esophageal striated muscle remains unknown. To better define the process of striated myogenesis, we examined myogenesis in murine fetal cultured esophageal whole-organ explants. Embryonic day 14.5 (E14.5) esophagi maintained a functional contractile phenotype for up to 7 days in culture. Striated myogenesis, as evidenced by myogenin expression, proceeded in a craniocaudal direction along the length of the esophagus. Esophageal length did not change during this process. Complete, but not partial, mechanical disruption of the rostral esophagus inhibited myogenesis distally. Addition of fibroblast growth factor-2 (FGF-2) to the culture media failed to inhibit striated myogenesis, but attenuated smooth muscle actin expression and reduced peristaltic activity. Inhibition of c-kit failed to inhibit peristalsis. These results suggest that striated myogenic precursors are resident along the entire length of the esophagus by day 14.5 and do not migrate along the esophagus after E14.5. Induction of myogenesis craniocaudally appears to require physical continuity of the esophagus and is not inhibited by FGF-2. Finally, peristalsis in E14.5 esophagi appears not to be regulated by interstitial cells of Cajal.

Beta2 and beta4 subunits of BK channels confer differential sensitivity to acute modulation by steroid hormones.

Membrane-associated receptors for rapid, steroidal neuromodulation remain elusive. Estradiol has been reported to facilitate activation of voltage- and Ca(2+)-dependent BK potassium channels encoded by Slo, if associated with beta1 subunits. We show here that 1) multiple members of the beta family confer sensitivity to multiple steroids on BK channels, 2) that beta subunits differentiate between steroids, and 3) that different betas have distinct relative preferences for particular steroids. Expressed in HEK 293 cells, inside-out patches with channels composed of Slo-alpha alone showed no steroid sensitivity. Cells expressing alphabeta4 exhibited potent, rapid, reversible, and dose-dependent potentiation by corticosterone (CORT; a glucocorticoid), and were potentiated to a lesser degree by other sex and stress steroids. In contrast, alphabeta2 channels were potentiated more strongly by dehydroepiandrosterone (DHEA; an enigmatic, stress-related adrenal androgen), and to a lesser extent by CORT, estradiol, testosterone, and DHEA-S. Cholesterol had no effect on any BK channel compositions tested. Conductance-voltage plots of channels composed of alpha plus beta2 or beta4 subunits were shifted in the negative direction by steroids, indicating greater activation at negative voltages. Thus our results argue that the variety of Slo-beta subunit coexpression patterns occurring in vivo expands the repertoire of Slo channel gating in yet another dimension not fully appreciated, rendering BK gating responsive to dynamic fluctuations in a multiple of steroid hormones.

Smooth muscle expression of Cre recombinase and eGFP in transgenic mice.

We report the generation of transgenic mice designed to facilitate the study of vascular and nonvascular smooth muscle biology in vivo. The smooth muscle myosin heavy chain (smMHC) promoter was used to direct expression of a bicistronic transgene consisting of Cre recombinase and enhanced green fluorescent protein (eGFP) coding sequences. Animals expressing the transgene display strong fluorescence confined to vascular and nonvascular smooth muscle. Enzymatic dissociation of smooth muscle yields viable, fluorescent cells that can be studied as single cells or sorted by FACS for gene expression studies. smMHC/Cre/eGFP mice were crossed with ROSA26/lacZ reporter mice to determine Cre recombinase activity; Cre recombinase was expressed in all smooth muscles in adult mice, and there was an excellent overlap between expression of the recombinase and eGFP. Initial smooth muscle-specific expression of fluorescence and Cre recombinase was detected on embryonic day 12.5. These mice will be useful to define smooth muscle gene function in vivo in mice, for the study of gene function in single, live cells, and for the determination of gene expression in vascular and nonvascular smooth muscle.

Regulation of human P2X1 promoter activity by beta helix-loop-helix factors in smooth muscle cells.

We isolated and characterized genomic clones of the human P2X1 receptor (hP2X1) gene in an effort to understand its tissue specific expression. The hP2X1 gene contains 12 exons spanning 20 kb, with exon sizes ranging from 59 to 143 bp. A 385 bp upstream fragment promoted hP2X1 gene expression in smooth muscle (A7R5 and primary trachealis) and fibroblast (NIH3T3) cell lines, and mutation of a consensus E box sequence (CACCTG) within this fragment (-340 to -345) did not alter basal promoter activity. However, co-transfected bHLH factors regulated activity of the 385 bp minimal P2X1 promoter in a tissue-specific manner. E12 expression inhibited and ITF2b augmented activity in A7R5 cells, but had no effect in NIH3T3 cells. ITF2a, Myo-D, and Id1 proteins had no effect on either cell line, but co-expression of ITF2a blocked E12 inhibition in A7R5 cells, while ITF2b failed to reverse the inhibition. Northern analysis of A7R5 RNA identified high levels of E12 and ITF2b transcripts, and gel shift assays using A7R5 and NIH3T3 nuclear extracts indicated the formation of a protein-DNA complex with an oligonucleotide corresponding to -330 and -348, which was abolished by base substitutions within the E box motif. Our results identify a critical E box response element in the hP2X1 promoter that binds bHLH factors and demonstrate smooth muscle specific transcriptional regulation by E proteins.

Guanylyl cyclase stimulatory coupling to K(Ca) channels.

We coexpressed the human large-conductance, calcium-activated K (K(Ca)) channel (alpha- and beta-subunits) and rat atrial natriuretic peptide (ANP) receptor genes in Xenopus oocytes to examine the mechanism of guanylyl cyclase stimulatory coupling to the channel. Exposure of oocytes to ANP stimulated whole cell K(Ca) currents by 21 +/- 3% (at 60 mV), without altering current kinetics. Similarly, spermine NONOate, a nitric oxide donor, increased K(Ca) currents (20 +/- 4% at 60 mV) in oocytes expressing the channel subunits alone. Stimulation of K(Ca) currents by ANP was inhibited in a concentration-dependent manner by a peptide inhibitor of cGMP-dependent protein kinase (PKG). Receptor/channel stimulatory coupling was not completely abolished by mutating the cAMP-dependent protein kinase phosphorylation site on the alpha-subunit (S869; Nars M, Dhulipals PD, Wang YX, and Kotlikoff MI. J Biol Chem 273: 14920-14924, 1998) or by mutating a neighboring consensus PKG site (S855), but mutation of both residues virtually abolished coupling. Spermine NONOate also failed to stimulate channels expressed from the double mutant cRNAs. These data indicate that nitric oxide donors stimulate K(Ca) channels through cGMP-dependent phosphorylation and that two serine residues (855 and 869) underlie this stimulatory coupling.

Hypoxia inhibits the Na(+)/Ca(2+) exchanger in pulmonary artery smooth muscle cells.

The cellular mechanisms underlying hypoxic pulmonary vasoconstriction are not fully understood. We examined the effect of hypoxia on Ca(2+) efflux from the cytosol in single Fura-2-loaded pulmonary artery myocytes. During mild hypoxia (pO(2)=50-60 Torr), peak [Ca(2+)](i) was increased and the rate of Ca(2+) removal from the cytosol was markedly slowed after stimuli that elevated [Ca(2+)](i). Removal of extracellular Na(+) potentiated the peak [Ca(2+)](i) rise and slowed the Ca(2+) decay rate in cells recorded under normoxic conditions; it did not further slow the Ca(2+) decay rate or potentiate the [Ca(2+)](i) increase in hypoxic cells. An Na(+)/Ca(2+) exchange current was recorded in isolated pulmonary artery myocytes. Switching from Li(+) to Na(+) (130 mM) revealed an inward current with reversal potential consistent with the Na(+)/Ca(2+) exchange current in cells in which [Ca(2+)](i) was clamped at 1 microM similar currents, although smaller, were observed with normal resting [Ca(2+)](i) using the perforated patch clamp technique. The Na(+)/Ca(2+) exchange current was markedly inhibited in myocytes exposed to mild hypoxia. RT-PCR revealed the expression of specific alternatively spliced RNAs of NCX1 in rat pulmonary arteries. These findings provide an enhanced understanding of the molecular mechanisms underlying hypoxic sensing in pulmonary arteries.

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release.

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.

HIV-1 gp120 and chemokines activate ion channels in primary macrophages through CCR5 and CXCR4 stimulation.

HIV type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry into target cells. Here we show that the HIV-1 envelope gp120 (Env) activates multiple ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. Env from both CCR5-dependent JRFL (R5) and CXCR4-dependent IIIB (X4) HIV-1 opened calcium-activated potassium (K(Ca)), chloride, and calcium-permeant nonselective cation channels in macrophages. These signals were mediated by CCR5 and CXCR4 because macrophages lacking CCR5 failed to respond to JRFL and an inhibitor of CXCR4 blocked ion current activation by IIIB. MIP-1beta and SDF-1alpha, chemokine ligands for CCR5 and CXCR4, respectively, also activated K(Ca) and Cl(-) currents in macrophages, but nonselective cation channel activation was unique to gp120. Intracellular Ca(2+) levels were also elevated by gp120. The patterns of activation mediated by CCR5 and CXCR4 were qualitatively similar but quantitatively distinct, as R5 Env activated the K(Ca) current more frequently, elicited Cl(-) currents that were approximately 2-fold greater in amplitude, and elevated intracellular Ca(+2) to higher peak and steady-state levels. Env from R5 and X4 primary isolates evoked similar current responses as the corresponding prototype strains. Thus, the interaction of HIV-1 gp120 with CCR5 or CXCR4 evokes complex and distinct signaling responses in primary macrophages, and gp120-evoked signals differ from those activated by the coreceptors' chemokine ligands. Intracellular signaling responses of macrophages to HIV-1 may modulate postentry steps of infection and cell functions apart from infection.

Signalling pathway for histamine activation of non-selective cation channels in equine tracheal myocytes.

1. The signalling pathway underlying histamine activation of non-selective cation channels was investigated in single equine tracheal myocytes. Application of histamine (100 microM) activated the transient calcium-activated chloride current (ICl(Ca)) and sustained, low amplitude non-selective cation current (ICat). The H1 receptor antagonist pyrilamine (10 microM) blocked activation of ICl(Ca) and ICat. Simultaneous application of histamine (100 microM) and caffeine (8 mM) during H1 receptor blockade activated ICl(Ca), but not ICat. Neither the H2 receptor antagonist cimetidine (20 microM) nor the H3 receptor antagonist thioperamide (20 microM) prevented activation of ICl(Ca) and ICat. 2. Intracellular dialysis of anti-Galphai/Galphao antibodies completely blocked activation of ICat by histamine, whereas ICl(Ca) was not affected. By contrast, anti-Galphaq/Galpha11 antibodies greatly inhibited ICl(Ca), but did not alter activation of ICat. 3. 1-Oleoyl-2-acetyl-sn-glycerol (OAG, 20-100 microM) did not induce any current or affect currents activated by histamine or methacholine (mACH). Simultaneous application of OAG and caffeine activated ICl(Ca), but not ICat, indicating that a rise in [Ca2+]i and stimulation of diacylglycerol-sensitive protein kinase C (PKC) is not sufficient to activate ICat. The phospholipase C inhibitor U73122 (2 microM) blocked histamine activation of ICl(Ca) and ICat, but simultaneous exposure of myocytes to histamine and caffeine restored both ICl(Ca) and ICat in the presence of U73122. 4. Histamine and mACH activated currents with equivalent I-V relationships. The currents activated by these agonists were not additive; following activation of ICat by mACH, histamine failed to induce an additional membrane current. Similarly, mACH did not induce an additional current after full activation of ICat by histamine. 5. We conclude that H1 histamine receptors activate ICat through coupling to Gi/Go proteins. Activation of ICat also requires intracellular calcium release, mediated by H1 receptors coupling to Gq/G11 proteins. This coupling is analogous to the activation of ICat by co-stimulation of M2 and M3 receptors.

Receptor avidity and costimulation specify the intracellular Ca2+ signaling pattern in CD4(+)CD8(+) thymocytes.

Thymocyte maturation is governed by antigen-T cell receptor (TCR) affinity and the extent of TCR aggregation. Signals provided by coactivating molecules such as CD4 and CD28 also influence the fate of immature thymocytes. The mechanism by which differences in antigen-TCR avidity encode unique maturational responses of lymphocytes and the influence of coactivating molecules on these signaling processes is not fully understood. To better understand the role of a key second messenger, calcium, in governing thymocyte maturation, we measured the intracellular free calcium concentration ([Ca2+]i) response to changes in TCR avidity and costimulation. We found that TCR stimulation initiates either amplitude- or frequency-encoded [Ca2+]i changes depending on (a) the maturation state of stimulated thymocytes, (b) the avidity of TCR interactions, and (c) the participation of specific coactivating molecules. Calcium signaling within immature but not mature thymocytes could be modulated by the avidity of CD3/CD4 engagement. Low avidity interactions induced biphasic calcium responses, whereas high avidity engagement initiated oscillatory calcium changes. Notably, CD28 participation converted the calcium response to low avidity receptor engagement from a biphasic to oscillatory pattern. These data suggest that calcium plays a central role in encoding the nature of the TCR signal received by thymocytes and, consequently, a role in thymic selection.

ATP-evoked Ca2+ transients and currents in murine thymocytes: possible role for P2X receptors in death by neglect.

The P2X family of ATP receptors (P2XR) have been implicated in thymocyte death in vitro and in vivo. We characterized ATP-evoked Ca2+ transients and membrane currents in thymocytes to better understand the role of P2XR during thymocyte development. ATP4-, but not UTP or GTP, activated a sustained non-selective cation current in voltage-clamped CD4- CD8- and CD4+ CD8+ thymocytes that was reversed by apyrase, which hydrolyzes ATP, and by the P2XR antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). The more selective P2XR agonist alphabeta-methylene ATP activated a smaller rapidly decaying current in both thymocyte populations. Reverse transcription-PCR results indicate that P2X1, P2X2, P2X6, and/or P2X7 are expressed in thymocytes. Finally, we used PPADS to examine the role of P2XR during thymocyte development in situ. PPADS-treated thymi yielded significantly more thymocytes (38%), due to a selective increase in CD4+ CD8+ cells. Together these data suggest that one or more PPADS-sensitive P2XR (P2X1, P2X2, P2X7) are involved in thymocyte apoptosis, and we propose more specifically a role associated with death by neglect.

Coupling of M2 muscarinic receptors to membrane ion channels via phosphoinositide 3-kinase gamma and atypical protein kinase C.

We report a novel signaling pathway linking M2 muscarinic receptors to metabotropic ion channels. Stimulation of heterologously expressed M2 receptors, but not other Gi/Go-associated receptors (M4 or alpha2c), activates a calcium- and voltage-independent chloride current in Xenopus oocytes. We show that the stimulatory pathway linking M2 receptors to these chloride channels consists of Gbeta gamma stimulation of phosphoinositide 3-kinase gamma (PI-3Kgamma), formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), and activation of atypical protein kinase C (PKC). The chloride current is activated in the absence of M2 receptor stimulation by the injection of PIP3, and PIP3 current activation is blocked by a pseudosubstrate inhibitory peptide of atypical PKC but not other PKCs. Moreover, the current is activated by injection of recombinant PKCzeta at concentrations as low as 1 nM. M2 receptor-current coupling was disrupted by inhibiton of PI-3K and by injection of beta gamma binding peptides, but it was not affected by expression of dominant negative p85 cRNA. We also show that this pathway mediates M2 receptor coupling to metabotropic nonselective cation channels in mammalian smooth muscle cells, thus demonstrating the broad relevance of this signaling cascade in neurotransmitter signaling.

M2 signaling in smooth muscle cells.

M2 receptor stimulation results in the gating of nonselective cation channels in several smooth muscle cell types. However the requirement for current activation includes a rise in cytosolic calcium mediated by M3 receptor induced calcium release. This complex signaling system confers substantial complexity on the interpretation of pharmacological experiments. M2 and M3 receptor stimulation has also been linked to the inhibition of potassium channels in smooth muscle. These signaling events are likely to play important roles in excitation/contraction coupling.

Cloning and characterization of the promoters of the maxiK channel alpha and beta subunits.

Large conductance, calcium-activated potassium (maxiK) channels are expressed in nerve, muscle, and other cell types and are important determinants of smooth muscle tone. To determine the mechanisms involved in the transcriptional regulation of maxiK channels, we characterized the promoter regions of the pore forming (alpha) and regulatory (beta) subunits of the human channel complex. Maximum promoter activity (up to 12.3-fold over control) occurred between nucleotides -567 and -220 for the alpha subunit (hSlo) gene. The minimal promoter is GC-rich with 5 Sp-1 binding sites and several TCC repeats. Other transcription factor-binding motifs, including c/EBP, NF-kB, PU.1, PEA-3, Myo-D, and E2A, were observed in the 5'-flanking sequence. Additionally, a CCTCCC sequence, which increases the transcriptional activity of the SM1/2 gene in smooth muscle, is located 27 bp upstream of the TATA-like sequence, a location identical to that found in the SM1/2 5'-flanking region. However, the promoter directed equivalent expression when transfected into smooth muscle and other cell types. Analysis of the hSlo beta subunit 5'-flanking region revealed a TATA box at position -77 and maximum promoter activity (up to 11.0-fold) in a 200 bp region upstream from the cap site. Binding sites for GATA-1, Myo-D, c-myb, Ets-1/Elk-1, Ap-1, and Ik-2 were identified within this sequence. Two CCTCCC elements are present in the hSlo beta subunit promoter, but tissue-specific transcriptional activity was not observed. The lack of tissue-specific promoter activity, particularly the finding of promoter activity in cells from tissues in which the maxiK gene is not expressed, suggests a complex channel regulatory mechanism for hSlo genes. Moreover, the lack of similarity of the promoters of the two genes suggests that regulation of coordinate expression of the subunits does not occur through equivalent cis-acting sequences.

Sodium hydrosulfite contractions of smooth muscle are calcium and myosin phosphorylation independent.

In an effort to further understand the processes underlying hypoxic pulmonary vasoconstriction, we examined the mechanism by which sodium hydrosulfite (Na2S2O4), a potent reducing agent and oxygen scavenger, induces smooth muscle contraction. In rat pulmonary arterial strips, sodium hydrosulfite (10 mM) induced contractions that were 65.9 +/- 12.8% of the response to 60 mM KCl (n = 9 segments). Contractions were not inhibited by nisoldipine (5 microM) or by repeated stimulation with caffeine (10 mM), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (10 microM), or cyclopiazonic acid (10 microM), all of which eliminated responses to contractile agonists. Maximum force generation after exposure to sodium hydrosulfite was 0.123 +/- 0.013 mN in the presence of 1.8 mM calcium and 0.127 +/- 0.015 mN in the absence of calcium. Sodium hydrosulfite contractions in pulmonary arterial segments were not due to the generation of H2O2 and occurred in the presence of chelerythrine (10 microM), which blocked phorbol ester contractions, and solution hyperoxygenation. Similar contractile responses were obtained in rat aortic and tracheal smooth muscles. Finally, contractions occurred in the complete absence of an increase in myosin light chain phosphorylation. Therefore sodium hydrosulfite-induced smooth muscle contraction is not specific to pulmonary arterial smooth muscle, is independent of calcium and myosin light chain phosphorylation, and is not mediated by either hypoxia or protein kinase C.

Calcium release and calcium-activated chloride channels in airway smooth muscle cells.

Rapid progress has been made in the determination of specific ion channels expressed in airway smooth muscle cells and their role in excitation-contraction coupling. The combination of molecular biology and molecular physiology has provided insight into the properties of voltage-dependent cation (calcium and potassium) channels and their regulation by excitatory and inhibitory signaling processes. In this brief review, we will focus on calcium release and calcium-activated chloride channels. The former channels mediate receptor-activated calcium release, and the latter channels are opened following this release event. Moreover, the discovery of spontaneous calcium release events, or "calcium sparks," in smooth muscle, suggests an unanticipated level of regulation. Intracellular calcium release can drive electrical activity by the activation of calcium-dependent sarcolemmal ion channels, including calcium-activated chloride channels. These channels activate briefly but undergo a rapid phosphorylation by calcium/calmodulin-dependent protein kinase, which uncouples channel activity from cytosolic calcium. The coupling between intracellular calcium release and depolarizing chloride currents represents a potentially important signaling system in airway smooth muscle.

Reconstitution of beta-adrenergic modulation of large conductance, calcium-activated potassium (maxi-K) channels in Xenopus oocytes. Identification of the camp-dependent protein kinase phosphorylation site.

The human large conductance, calcium-activated potassium (maxi-K) channel (alpha and beta subunits) and beta2-adrenergic receptor genes were coexpressed in Xenopus oocytes in order to study the mechanism of beta-adrenergic modulation of channel function. Isoproterenol and forskolin increased maxi-K potassium channel currents in voltage-clamped oocytes expressing the receptor and both channel subunits by 33 +/- 5% and 35 +/- 8%, respectively, without affecting current activation or inactivation. The percentage of stimulation by isoproterenol and forskolin was not different in oocytes coexpressing the alpha and beta subunits versus those expressing the only the alpha subunit, suggesting that the alpha subunit is the target for regulation. The stimulatory effect of isoproterenol was almost completely blocked by intracellular injection of the cyclic AMP dependent protein kinase (cAMP-PK) regulatory subunit, whereas injection of a cyclic GMP dependent protein kinase inhibitory peptide had little effect, indicating that cellular coupling of beta2-adrenergic receptors to maxi-K channels involves endogenous cAMP-PK. Mutation of one of several potential consensus cAMP-PK phosphorylation sites (serine 869) on the alpha subunit almost completely inhibited beta-adrenergic receptor/channel stimulatory coupling, whereas forskolin still stimulated currents moderately (16 +/- 4%). These data demonstrate that physiological coupling between beta2 receptors and maxi-K channels occurs by the cAMP-PK mediated phosphorylation of serine 869 on the alpha subunit on the channel.

The human P2X4 receptor gene is alternatively spliced.

ATP acts as a fast excitatory neurotransmitter by binding to a large family of membrane proteins, P2X receptors, that have been shown to be ligand-gated, non-selective cation channels. We report the cloning of a full-length and alternatively spliced form of the human P2X4 gene. Clones were identified from a human stomach cDNA library using a rat P2X4 probe. Nucleotide sequence analysis of positive clones identified the full-length human P2X4 cDNA, which codes for a 388-residue protein that is highly homologous (82%) to the rat gene, and an alternatively spliced cDNA. In the alternatively spliced cDNA, the 5'-untranslated region and the first 90 amino acids in the coding region of full-length human P2X4 are replaced by a 35 amino acid coding sequence that is highly homologous with a region of chaperonin proteins in the hsp-90 family. The open reading frames of the full-length and splice variant clones were confirmed by in vitro translation. Northern analysis indicated expression of the full-length P2X4 message in numerous human tissues including smooth muscle, heart, and skeletal muscles. Alternatively spliced RNAs were identified in smooth muscle and brain by RT-PCR and confirmed by RNAse protection assays using a 710 bp anti-sense RNA probe that spanned the alternatively spliced and native P2X4 regions. Injection of full-length, but not alternatively spliced, cRNA into Xenopus oocytes resulted in the expression of ATP gated non-selective cation currents.