GABA is the most important CNS inhibitory neurotransmitter. GABAA receptors (GABARs) are composed of subtypes from six families (alphal-6, beta1-3, gamma1-3, delta, epsilon, pi). Cytoplasmic loops between the 3rd and 4th transmembrane domains of several subtypes contain phosphorylation consensus sequences. Protein kinase A (PKA) and C (PKC) variably modified GABAR current in different neuronal and recombinant preparations. Effects of GABAR phosphorylation in neurons are uncertain since multiple undetermined GABAR isoforms are present in neurons, and biochemical studies of specific cell type GABARs has not been possible since pure populations of specific neurons cannot be obtained. To overcome these problems, we propose to use human stem (NT2) and neuronal (NT2-N) cell lines and recombinant GABARs expressed in mammalian cells. The hypotheses to be tested are: 1) Activation of PKA or PKC enhances NT2-N neuronal and NT2 precursor cell GABAR currents. 2) Phosphorylation of a single beta subunit subtype serine (S409 in the beta1, S410 in the beta2 and S408 in the beta3 subtype) by PKA or PKC enhances recombinant alphax-betay-gamma3 GABAR currents (x = 2, 3 or 5; y = 1, 2 or 3). 3) Phosphorylation of GABARs on a single beta subtype serine (S408 in the beta3 subtype) by PKA or PKC produces enhancement of NT2-N neuronal and NT2 precursor cell GABAR current. 4) Phosphorylation of GABAR channels by PKA or PKC enhances GABAR current by modifying rates of entry into and/or out of desensitized states, thus altering the time course of GABAR currents.
The specific aims are to determine: 1) The GABAR subtype proteins in NT2 and NT2-N cells. 2) The pharmacological, steady state single channel kinetic and transient kinetic properties of """"""""NT2 and NT2-N"""""""" GABARs expressed in mammalian fibroblasts. 3) The functional consequences of activation of PKA and PKC on recombinant alpha-betax-gamma3 GABAR currents recorded from mammalian cells. 4) The effects of phosphorylation of beta2 and beta3 subtype serines on alpha-betax-gamma3 GABAR currents. S) The biophysical mechanisms of PKA and PKC modification of recombinant GABAR currents. 6) The functional consequences of activation of PKA and PKC on GABAR currents in NT2 and NT2-N cells. 7) The NT2 and NT2-N GABAR subtype protein phosphorylated by PKA and PKC. 8) The biophysical mechanisms PKA and PKC modification of NT2 and NT2-N GABAR currents.
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