Functional properties of heteromeric P2X_1/5 receptors expressed in HEK cells and excitatory junction potentials in guinea-pig submucosal arterioles

Abstract

P2X receptors are ATP-gated cation channels; they form as homomers or heteromers from a family of seven related subunits. In particular, heteromeric channels comprising P2X₂ and P2X₃ subunits, or P2X₁ and P2X₅ subunits, show distinctive physiological and pharmacological properties in heterologous expression systems. There is substantial evidence that one of the native P2X receptors in sensory neurones corresponds to the P2X_2/3 heteromer, but there is no evidence for P2X_1/5 heteromers in native tissue. We recorded currents in response to activation of heteromeric P2X_1/5 receptors expressed in HEK293 cells to characterize further their functional properties. The ATP concentration–response curve had a threshold concentration of 1 nM, and a Hill slope of one. TNP-ATP was a weak partial agonist, and a non-competitive antagonist which inhibited maximal ATP currents by 60%. Increasing or decreasing pH from 7.3 shifted the ATP concentration–response curves to the right by fivefold and decreased the maximum current by 40%. Calcium permeability was lower than that observed for other P2X receptors (P_Ca/P_Na ratio=1.1). The nanomolar sensitivity of this receptor revealed a steady release of ATP from HEK293 cells, providing an extracellular concentration which ranged from 3 to 300 nM. Noradrenaline (0.3–30 μM) increased ATP-evoked currents by 35%; this facilitation occurred within 20 ms. We also recorded excitatory junction potentials (EJPs) from guinea-pig submucosal arterioles. EJPs were inhibited by suramin and PPADS (IC₅₀s of 0.2 μM and 20 μM) but TNP-ATP (0.1–10 μM) inhibited EJPs by <30%. Noradrenaline (0.3–30 μM in the presence of phentolamine and propranolol) decreased EJPs in control preparations but facilitated EJPs by 5–20% in submucosal arterioles from reserpinized guinea-pigs. These properties are discussed in relation to P2X receptors underlying EJPs at autonomic neuroeffector junctions.

Introduction

Burnstock and Holman (1961) first described excitatory junction potentials (EJPs) at autonomic neuroeffector junctions. In these pioneering studies, they showed clearly that the EJPs and subsequent contraction of the smooth muscle in the vas deferens resulted from neurotransmitter released from sympathetic nerves. At that time, perhaps influenced by von Euler’s Nobel-winning discovery of noradrenaline as the sympathetic transmitter (von Euler, 1951), they concluded that the EJPs were due to the release of noradrenaline on to adrenoceptors, even though they also noted that EJPs were resistant to blockade by the adrenergic (or cholinergic) antagonists then available (Burnstock and Holman, 1963). The notion that EJPs in vascular and visceral smooth muscle resulted from activation of atypical adrenoceptors held sway for most of the next three decades, but Burnstock and colleagues were accumulating much evidence for ATP as the transmitter responsible for EJPs in many smooth muscles (Burnstock, 1972, Burnstock, 1976, Burnstock, 1990). Burnstock termed these receptors for ATP the P2X receptors (Burnstock and Kennedy, 1985). The identification of reasonably selective P2X receptor agonists and antagonists in the late 1980s and early 1990s (Burnstock, 1990), the molecular cloning of the P2X receptors in 1994–1996 (Valera et al., 1994, Brake et al., 1994; North and Barnard, 1997), and the generation of transgenic mice lacking both P2X₁ receptors and EJPs in 2000 (Mulryan et al., 2000) now confirm Burnstock’s original proposals of ATP as a major neurotransmitter in the autonomic nervous system.

The P2X₁ receptor was first identified by expression cloning methods using a vas deferens cDNA library (Valera et al., 1994). Subsequent immunohistochemical localization with P2X₁-specific antibodies revealed dense staining in smooth muscle of the vas deferens and most peripheral vascular beds (Vulchanova et al., 1996). Electrophysiological studies in heterologous expression systems (oocytes or human embryonic kidney (HEK) 293 cells) have shown that the pharmacological and physiological properties of cloned P2X₁ receptors are very similar to those of native P2X receptors in isolated single cells of vas deferens or arterial smooth muscle. The similarities include agonist and antagonist sensitivities, onset kinetics, the desensitization during agonist application, the marked decline in the response with repeated agonist applications, high calcium permeability, pH and divalent cation dependence, and single-channel conductance (Benham and Tsien, 1987, Evans and Kennedy, 1994, Valera et al., 1994, Evans et al., 1995, Evans et al., 1996, Khakh et al., 1995, Trezise et al., 1995, Evans, 1996, Stoop et al., 1997; see Evans et al., 1997). This is all quite consistent with the conclusion that native P2X receptors in isolated smooth muscle are formed by homomeric assembly of P2X₁ subunits.

Nevertheless, there are striking discrepancies between the properties of EJPs and ATP-evoked contractions in smooth muscle and P2X₁-mediated responses in isolated cells. EJPs, as well as the underlying excitatory junction currents (EJCs) show strong facilitation with increasing frequency of stimulation (Burnstock and Holman, 1963, Bennett, 1972, Bennett, 1996, Hirst, 1977, Holman and Surprenant, 1979, Surprenant, 1980, Cunnane and Stjärne, 1982, Brock and Cunnane, 1988), whereas currents in cells expressing P2X₁ receptors show marked rundown in responses to repeated brief applications (0.2–1 s) of ATP (Valera et al., 1994, Evans et al., 1995, Evans et al., 1997). Moreover, when ATP is superfused onto in vitro preparations of small arteries and arterioles, depolarizations and vasoconstrictions both have initial rapid transients and subsequent sustained components (Burnstock, 1988, Evans and Surprenant, 1992, Galligan et al., 1995); the responses of cells expressing P2X₁ receptors undergoes complete desensitization within 1 to 3 s (see Evans et al., 1997). 2′3′-O-(2,4,6-Trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) blocks expressed P2X₁ receptors at low nanomolar concentrations (Virginio et al., 1998) but is ineffective in blocking α,β-methylene-ATP (αβmATP)-induced contractions (Lewis et al., 1998); this ineffectiveness might be attributed to limited access, or degradation, of the TNP-ATP in the intact tissues as compared to isolated cells (Lewis et al., 1998). Alternatively, one interpretation of these discrepancies is that P2X receptors activated by sympathetically released ATP at the neuroeffector junction might not be homomeric P2X₁ receptors.

P2X₁ and P2X₅ receptors heteropolymerize to form an ATP-gated cation channel whose properties resemble in three important respects those of EJPs in vascular smooth muscle: they show calcium-dependent facilitation upon application of ATP, they show both transient and sustained components and they are less sensitive to inhibition by TNP-ATP than are P2X₁ receptors (Torres et al., 1998, Lê et al., 1999, Haines et al., 1999). Low levels of P2X₅ mRNA have been detected in vascular smooth muscle (Phillips and Hill, 1999) and immunostaining of other autonomic tissue has been detected with P2X₅-selective antibodies (Meyer et al., 1999, Afework and Burnstock, 1999). The aim of the present study was to provide further characterization of the pharmacological and physiological properties of heteromeric P2X_1/5 receptors expressed in HEK293 cells with a view to assessing their potential involvement at autonomic neuroeffector junctions. Toward this aim we also recorded purinergic contractions and neurogenic EJPs in the submucosal arteriolar preparation — a ‘reduced’ preparation in which nucleotide degradation and drug access barriers are expected to be minimal (see Hirst, 1989).