Functional α6-Containing Nicotinic Receptors Are Present in Chick Retina

Abstract

Despite the fact that the neuronal chick alpha6 subunit was first cloned several years ago and recently has been shown to form acetylcholine (ACh)-activated channels in heterologous systems, no information is yet available concerning the structure and function of the alpha6-containing nicotinic receptors in neuronal tissues. Using subunit-specific antibodies directed against two different epitopes of the chick alpha6 subunit, we performed immunoprecipitation experiments on immunopurified alpha6-containing receptors radiolabeled with the nicotinic agonist [3H]epibatidine (Epi): almost all of the alpha6 receptors contained the beta4 subunit, 51% the beta3 subunit, 42% the alpha3 subunit, and 7.5% the beta2 subunit. Western blot analyses of the purified receptors confirmed the presence of the alpha3, beta3, beta2, and beta4 subunits, and the absence of the alpha4, alpha5, and alpha7 subunits. The alpha6-containing receptors bind [3H]Epi (Kd = 35 pM) and a number of other nicotinic agonists with very high affinity, the rank order being Epi >> cytisine > nicotine > 1, 1-dimethyl-4-phenylpiperazinium > acetylcholine > carbamylcholine. The alpha6 receptors also have a distinct antagonist pharmacological profile with a rank order of potency of alpha-conotoxin MII > methyllycaconitine > dihydro-beta-erythroydine > MG624 > d-tubocurarine > decamethonium > hexamethonium. When reconstituted in lipid bilayers, the alpha6-containing receptors form functional cationic channels with a main conductance state of 48 pS. These channels are activated by nicotinic agonists in a dose-dependent manner, and blocked by the nicotinic antagonist d-tubocurarine.

Synaptically Released and Exogenous ACh Activates Different Nicotinic Receptors to Enhance Evoked Glutamatergic Transmission in the Lateral Geniculate Nucleus

“…Immunoimmobilized chick ␣6* receptors exhibited the following K i s against 3 H-epibatidine binding: ␣-CTx MII, 66 nM; MLA, 1.35 M, DH␤E, 2.8 M (Vailati et al 1999). In comparison, the concentrations required to completely block the functional response to conditioning stimulation in this study were as follows: ␣-CTx MII, 100 nM; MLA, 1.0 M, DH␤E, 0.1 M. It is known that the ␣6-containing receptors in retinal ganglion cells are heterogeneous, and many also contain ␣3 and/or ␤3 subunits (Vailati et al 1999(Vailati et al , 2000(Vailati et al , 2003. In both chick and rodent, these ␣6* receptors migrate down the optic nerve and tract and are present in the optic lobe and LGNv (Champtiaux et al 2002;Vailati et al 1999).…”

“…Instead, the response to endogenously released ACh seems to be mediated primarily by presynaptic ␣6-and/or ␣3-containing nAChRs based on pharmacological properties (Mogg et al 2002;Salminen et al 2004). Immunoimmobilized chick ␣6* receptors exhibited the following K i s against 3 H-epibatidine binding: ␣-CTx MII, 66 nM; MLA, 1.35 M, DH␤E, 2.8 M (Vailati et al 1999). In comparison, the concentrations required to completely block the functional response to conditioning stimulation in this study were as follows: ␣-CTx MII, 100 nM; MLA, 1.0 M, DH␤E, 0.1 M. It is known that the ␣6-containing receptors in retinal ganglion cells are heterogeneous, and many also contain ␣3 and/or ␤3 subunits (Vailati et al 1999(Vailati et al , 2000(Vailati et al , 2003.…”

“…In comparison, the concentrations required to completely block the functional response to conditioning stimulation in this study were as follows: ␣-CTx MII, 100 nM; MLA, 1.0 M, DH␤E, 0.1 M. It is known that the ␣6-containing receptors in retinal ganglion cells are heterogeneous, and many also contain ␣3 and/or ␤3 subunits (Vailati et al 1999(Vailati et al , 2000(Vailati et al , 2003. In both chick and rodent, these ␣6* receptors migrate down the optic nerve and tract and are present in the optic lobe and LGNv (Champtiaux et al 2002;Vailati et al 1999). Thus the most parsimonious explanation for the enhancement of glutamatergic transmission observed in this study after conditioned stimulation is that ACh released from cholinergic fibers activates ␣6* and/or ␣3* (␣6*/␣3*) nAChRs located on retinal ganglion nerve terminals, causing enhanced glutamate release.…”

“…The ␣6 and ␤3 nAChR subunits are not widely distributed in neuronal tissue, but both are expressed in the retina of chick and mice (Champtiaux et al 2002;Vailati et al 1999Vailati et al , 2000. In the chick, two nAChR subtypes containing ␤3 subunits have been immunopurified and pharmacologically characterized, one of which also contains at least one ␣6 subunit (Vailati et al 2000).…”

The role of nAChR‐mediated spontaneous retinal activity in visual system development

“…Extensive work using immunohistochemistry and in situ hybridization in chick, mouse, rat, and rabbit have identified several subunits of nAChRs in the retina. There is expression of several subunits that comprise heteromeric nAChRs in the IPL, including the ␣3-6 (and ␣8 in chick) and ␤2-4 subunits (Swanson et al, 1987;Hoover and Goldman, 1992;Keyser et al, 1993;Hamassaki-Britto et al, 1994;Cimino et al, 1995;Zoli et al, 1995;Vailati et al, 1999;Champtiaux et al, 2002). Both mRNA and protein have been localized to several cell types in the inner retina, including RGCs, amacrine cells, displaced amacrine cells, and bipolar cells.…”