This is a renewal of a project going into its 19th year, which was last reviewed in 1986. The applicant has isolated clones of several alpha and beta subunits of neuronal nicotinic receptors, sequenced these clones, determined anatomical distribution of the subunits in the CNS, expressed clones in frog oocytes, and described channel functions for most of the subunits when expressed as alpha/beta combinations or as single subunits in the case of the alpha7 subunit. The applicant now proposes to use these nucleic acid probes, and antibodies which he has generated, to examine a series of issues relating to functional receptor composition, assembly, disposition on the cell surface, and role in synapse formation. The first specific aim will involve the development of a transient expression system in mammalian cells which will allow various subunit combinations to be expressed in order to examine issues of subunit pairing, function (particularly as related to calcium permeability) and targeting of subunits to particular cell surfaces. Particular interest is given in the proposal to the alpha7 subunit because of several interesting properties it demonstrates including inhibition by bungarotoxin, the ability to produce in oocytes a functional channel by itself, and the high calcium permeability of the homo-oligomeric channel.
Specific Aim 2 will examine the anatomical distribution of alpha7 on cultured neurons, the calcium permeabilities associated with expected areas of alpha7 subunits, and the role of alpha7 activation on neurite outgrowth. Another observation made with the alpha7 subunit is that the ability of the subunit, by itself, to produce a functional channel in oocytes is prevented by exposure of the cells to the peptidyl-prolyl isomerase inhibitor cyclosporin A, thus implicating a role for this enzyme in functional receptor production.
In Aim 3 the applicants will test the hypothesis that this enzyme is involved in homo- oligomeric receptors such as the alpha7 receptor. This will be approached through studies examining direct physical association of the enzyme with the subunit, by studies involving mutagenesis of the prolines in alpha7 and other subunits, and through functional studies of receptors formed in oocytes and transfected cells.
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