Properties of native P2X receptors in rat trigeminal mesencephalic nucleus neurones: lack of correlation with known, heterologously expressed P2X receptors

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Abstract

Trigeminal mesencephalic nucleus (MNV) neurones express functional P2X receptors. In order to determine the molecular identity of the P2X receptors in this nucleus we have used whole cell patch clamp recording of P2X receptor-mediated currents to determine the pharmacological properties of the receptors, and have compared them with those of cloned P2X receptor subunits. The purine nucleotides ATP (300 μM), ATP-γ-S (30 μM) and αβmeATP (300 μM) evoked inward currents in all MNV neurones whereas αβmeADP (300 μM) did not. βγme-L-ATP (300 μM) evoked only a small (∼20 pA) current in 3 out of 6 MNV neurones. The P2X receptor antagonist TNP–ATP (10 nM–10 μM) and raised extracellular Ca2+ (8 and 30 mM) reduced, but did not abolish, the current evoked by ATP-γ-S. The current remaining in TNP–ATP was insensitive to blockade by raised Ca2+. These properties suggest that MNV neurones do not express homomeric P2X3, P2X4 or P2X6 receptors. Whilst the TNP–ATP-insensitive ATP-γ-S-evoked current has many characteristics similar to both homomeric P2X2 and P2X5 receptors, its insensitivity to blockade by raised Ca2+ is difficult to reconcile with the receptor being a P2X2 or P2X5 homomeric channel. More likely, the receptor is a heteromer that comprises either or both of these subunits. The TNP–ATP-sensitive component of the ATP-γ-S-evoked current is dissimilar to known cloned homomeric or heteromeric P2X receptors.

Introduction

P2X receptors are ATP-gated cation channels found on many types of excitable cell including peripheral and central neurones (see North and Barnard, 1997 for review). Extracellular ATP, acting through P2X receptors, has been shown to mediate both fast synaptic transmission (Edwards et al., 1992, Evans et al., 1992, Silinsky et al., 1992, Galligan and Bertrand, 1994, Bardoni et al., 1997, Nieber et al., 1997, Jo and Schlichter, 1999) and presynaptic modulation of neurotransmitter release (Gu and MacDermott, 1997, Khakh and Henderson, 1998, Boehm, 1999). Over the last 5 years much effort has been devoted to understanding the identity of P2X receptors. To date seven P2X receptor subunit genes (P2X1–P2X7) and several splice variants have been cloned (for reviews see Evans and Surprenant, 1996, North, 1996, North and Barnard, 1997). When heterologously expressed all of these subunits form homomeric channels and some have been shown to form functional heteromeric channels (P2X2/3, P2X4/6 and P2X1/5) that differ with regard to agonist and antagonist selectivity, desensitisation and single channel properties (Evans et al., 1995, Lewis et al., 1995, Buell et al., 1996, Collo et al., 1996, Evans, 1996, Michel et al., 1996, Le et al., 1998, Torres et al., 1998, Haines et al., 1999, Le et al., 1999). Knowledge of these properties of cloned channels has been used to determine the composition of P2X receptors in some sensory neurones (Chen et al., 1995, Lewis et al., 1995, Robertson et al., 1996, Cook et al., 1997, Rae et al., 1998, Thomas et al., 1998, Grubb and Evans, 1999).

For several reasons the properties of the cloned channels may not exactly match those observed for native receptors. First, new heteromeric complexes of known P2X receptor subunits may be assembled. Indeed, recent biochemical studies indicate that many different subunits are able to coassemble to form heteromeric complexes in heterologous expression systems (Torres et al., 1999). Second, other, as yet still to be cloned P2X receptor subunits or novel splice variants of known subunits may exist. Third, native P2X receptor subunits could possibly co-assemble with either accessory subunits or subunits from other ligand gated ion channels (Zhou and Galligan, 1998, Barajas-López et al., 1998, Searl and Silinsky, 1998). Fourth, native receptors may be modified by regulatory pathways that are absent in heterologous expression systems. Therefore, it is important to determine if the properties of endogenous P2X receptors are similar to, or different from, those of heterologously expressed P2X receptors.

The trigeminal mesencephalic nucleus (MNV) contains cell bodies of primary afferent, sensory neurones that relay proprioceptive information from the teeth, periodontium, hard plate and joint muscles. In situ hybridisation experiments have detected mRNA for P2X2 (Kanjhan et al., 1999), P2X4, P2X5 and P2X6 (Collo et al., 1996) subunits in MNV neurones and we and others have previously demonstrated that these neurones express functional P2X receptors (Cook et al., 1997, Khakh et al., 1997). In the present study we have sought to characterise further the type(s) of P2X receptor(s) present on the soma of MNV neurones. We compare our data on the properties of endogenously expressed P2X receptors in MNV neurones contained in brain slices with those previously reported for cloned P2X channel subunits.

Section snippets

Brain slices

Pontine brain slices containing the MNV and locus coeruleus (LC) were prepared as described previously (Henderson et al., 1982). Briefly, male Wistar rats (both young, 14–28 days postnatal, and mature rats, weight 220–240 g) were humanely killed by stunning and cervical dislocation. The scalp and skull were removed and the brain excised. A block of tissue containing the pons and cerebellum was prepared and mounted on a glass slide, rostral surface uppermost, using cyanoacrylate glue. Slices

Effects of purine nucleotides on MNV neurones

ATP and its analogues ATP-γ-S, αβmeATP, βγmeATP, βγme-L-ATP, and αβmeADP were tested on MNV neurones from young rats. As previously reported ATP, ATP-γ-S, and αβmeATP evoked inward currents associated with an increase in membrane conductance that desensitised in the continued presence of the agonist (Fig. 1) (Khakh et al., 1997). The low potency of ATP on MNV neurones is probably because ATP is rapidly degraded in the brain slice preparation. However, αβmeATP is more resistant to degradation (

Discussion

Functional P2X receptors have previously been demonstrated on both the soma and terminals of MNV neurones (Cook et al., 1997, Khakh et al., 1997, Khakh and Henderson, 1998). The purpose of the present study was to characterise more fully the type(s) of native P2X receptors expressed on the somata of MNV neurones. The data we have obtained can be compared with those currently available from studies of other natively expressed P2X receptors on other types of neurone and to studies of cloned

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  • Cited by (0)

    1

    Present address: Parke-Davis Neuroscience Research Centre, Cambridge CB2 2QB, UK.

    2

    Present address: Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125, USA.

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