Modulation of the Ca²⁺ conductance of nicotinic acetylcholine receptors by Lypd6

Abstract

The agonist binding sensitivity and desensitization kinetics of nicotinic acetylcholine receptors (nAChRs) can be modulated by snake venom neurotoxins and related endogenous small proteins of the uPAR-Ly6 family. Here we identify Lypd6, a distantly related member of the u-PAR/Ly-6 family expressed in neurons as a novel modulator of nAChRs. Lypd6 overexpressed in trigeminal ganglia neurons selectively enhanced the Ca²⁺-component of nicotine-evoked currents through nAChRs, as evidenced by comparative whole-cell patch clamp recordings and Ca²⁺-imaging in wildtype and transgenic mice overexpressing Lypd6. In contrast, a knockdown of Lypd6 expression using siRNAs selectively reduced nicotine-evoked Ca²⁺-currents. Pharmacological experiments revealed that the nAChRs involved in this process are heteromers. Transgenic mice displayed behaviors that were indicative of an enhanced cholinergic tone, such as a higher locomotor arousal, increased prepulse-inhibition and hypoalgesia. These mice overexpressing Lypd6 mice were also more sensitive to the analgesic effects of nicotine. Transgenic mice expressing siRNAs directed against Lypd6 were unable to procreate, thus indicating a vital role for this protein. Taken together, Lypd6 seems to constitute a novel modulator of nAChRs that affects receptor function by selectively increasing Ca²⁺-influx through this ion channels.

Introduction

Nicotinic acetylcholine receptors (nAChRs) belong to the “Cys-loop superfamily” of ligand-gated ion channels that are gated by the neurotransmitter acetylcholine, as well as nicotine, the major psychoactive component in tobacco. They have an important role in various functions of the central nervous system (CNS) including memory, locomotion, sensorimotor gating and pain sensation (Dani, 2001, Everitt and Robbins, 1997, Picciotto et al., 2000, Tassonyi et al., 2002). Based upon their expression and subunit composition, nAChRs can have quite different effects on neurotransmission. Presynaptic and preterminal nAChRs enhance neurotransmitter release (Lena et al., 1993, McGehee et al., 1995), postsynaptic nAChRs convey fast excitatory transmission (Hefft et al., 1999, Roerig et al., 1997) and nonsynaptic nAChRs have effects on many neurotransmitter systems by modulating neuronal excitability (Descarries et al., 1997, Vizi, 2000). In these processes influx of Ca²⁺ through nAChR-channels plays an important functional role (Dani, 2001, Vizi, 2000).

Nicotinic acetylcholine receptors represent a prototype of allosteric proteins. In addition to modulation through allosteric effectors such as divalent cations, natural steroid hormones (Curtis et al., 2002), drugs like 5-hydroxyindole (Zwart et al., 2002), PNU-120596 (Hurst et al., 2005) or ivermectin (Krause et al., 1998), several endogenous small proteins with homology to alpha-neurotoxins have been identified that bind nAChRs and modulate their activity (Hogg et al., 2005).

On neurons, the GPI-anchored protein Lynx1 alters current amplitudes and desensitization kinetics of α4/β2-nAChRs (Ibanez-Tallon et al., 2002, Miwa et al., 1999). Genetic deletion of Lynx1 in mice resulted in an increased sensitivity of nAChRs to nicotine and a prolonged nAChR receptor activation (Miwa et al., 2006). In the epidermis, the secreted proteins Slurp1 and Slurp2 regulate keratinocyte nAChRs (Adermann et al., 1999, Arredondo et al., 2006, Arredondo et al., 2005). Mutations in the human SLURP1 gene cause Mal de Meleda, an autosomal recessive inflammatory and palmoplantar skin disorder (Chimienti et al., 2003). Lynx1, Slurp1 and Slurp2 belong to the murine u-PAR/Ly-6 gene family, which is evolutionary related to the elapid snake venom neurotoxins (Fleming et al., 1993, Gumley et al., 1995). Most other members of the u-PAR/Ly-6 gene family code for GPI-linked cell accessory proteins of the immune system (Gumley et al., 1995, Hanninen et al., 1997, Mallya et al., 2006).

In this study we report the identification of a novel member of the u-PAR/Ly6 gene family, Lypd6 (LY6/PLAUR domain containing 6 (Ploug and Ellis, 1994)), which modulates nicotinic receptor signaling in vivo. Sequence analysis of Lypd6 and other members of the snake venom neurotoxin superfamily support a direct evolutionary relationship between Lypd6 and well-known endogenous modulators of cholinergic transmission like Lynx1, Slurp1 and Slurp2. Lypd6 is highly expressed in the brain and spinal cord. Lypd6-deficient and overexpressing transgenic mice reveal that Lypd6 acts as a modulator of nAChRs and is also essential for embryo development.