The overall goal of this proposal is to identify molecular mechanisms that regulate potassium channel function and its contribution to electrical excitability during initial stages of amphibian spinal neuron differentiation. Our previous studies indicate that by the time amphibian spinal neurons begin to form synapses, the density of voltage-dependent delayed rectifier-type potassium current (IKv) is maintained within narrow limits. We refer to this tightly regulated level of potassium current as its set point. Voltage dependent potassium (Kv) channels are multimeric proteins consisting of pore-forming alpha subunits (e.g., Kv1- Kv4) and auxiliary beta subunits (Kv-beta1-Kv-beta4). Our previous work has demonstrated that Kv2alpha and Kv1alpha subunits contribute to the IKv of spinal neurons and that Kv-beta subunits are co expressed with Kv2alpha and Kv1alpha subunits in the developing spinal cord. The 3 Specific Aims of this proposal focus on the mechanisms that regulate the density of functional Kv2 and Kvl channel complexes and achieve homeostasis of potassium current in mature neurons. We propose that the numbers of functional Kv2 and Kv1 channels in spinal neurons are regulated by different mechanisms. For Kv2 channels, we hypothesize that post-translational mechanisms that target the cytoplasmic proximal carboxyl-terminus (proxC) regulate functional expression of Kv2.2alpha subunits (Aims 1 and 2). In contrast, for Kv1 channels, we propose that transcriptional mechanisms play a major role by limiting the amount of Kv1alpha subunits. However, our data suggest that Kv-beta2 subunits are a necessary component of myocyte Kv1 channel complexes in vivo. Here, we propose to test this possibility for spinal neurons (Aim 3). A combination of embryological, electrophysiological, molecular biological and immunological techniques will be used to fulfill the two Aims. The experimental preparation -the Xenopus embryo - provides an ideal system for analysis of post-transcriptional mechanisms in embryonic neurons, because manipulation of RNA levels is easily achieved by microinjection of RNA into early cleavage stage embryos. Because electrical activity plays key roles during neuronal development including regulation of process outgrowth, synapse formation and patterns of gone expression, the information provided by these studies will allow us to define the role of electrical excitability in differentiation and function of the emerging nervous system.
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