Date of Award

2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Neuroscience

First Advisor

Morielli, Anthony

Abstract

The voltage-gated potassium channel Kv1.2 is a critical modulator of neuronal physiology, including dendritic excitability, action potential propagation, and neurotransmitter release. However, mechanisms by which Kv1.2 may be regulated in the brain are poorly understood. In heterologous expression systems Kv1.2 is regulated by endocytosis of the channel from the plasma membrane, and this trafficking can be modulated by adenylate cyclase (AC). The goal of this dissertation was to determine whether AC modulated endocytic trafficking of endogenous Kv1.2 occurred in the mammalian nervous system. Within the brain, Kv1.2 is expressed at its highest levels in the cerebellar cortex. Specifically, Kv1.2 is expressed in dendrites of Purkinje cells (PC), the sole efferent neurons of the cerebellar cortex; Kv1.2 is also expressed in axon terminals of Basket cells (BC), which make inhibitory synapses to Purkinje cells. The loss of functional Kv1.2 in PC dendrites or BC axon terminals causes profound changes in the neurophysiology of Purkinje cells, and aberrant loss of Kv1.2 produces cerebellar ataxia. Therefore, the cerebellum offers a brain structure where Kv1.2 is abundant and has known and important roles in synaptic physiology. A candidate regulator of Kv1.2 trafficking in cerebellar synapses is the secretin peptide receptor: the receptor is also located in both PC dendrites and BC axon terminals, and ligand binding to the secretin receptor stimulates AC. Although secretin affects cerebellar neurophysiology and cerebellar dependent behavior, the mechanisms are not well resolved. By cell-surface protein biotinylation and subsequent immunoblot quantitation of secretin treated rat cerebellar slice lysates, secretin was found to decrease cell-surface Kv1.2. This effect could be mimicked by stimulating AC with forskolin, and could be occluded by inhibition of the secretin receptor, AC, or protein kinase A. The secretin receptor stimulated loss of surface Kv1.2 was not accompanied by decreased total Kv1.2 protein levels, but did involve enhanced channel endocytosis. Microscopy studies using two novel independent techniques provided evidence that both BC axon terminals and PC dendrites are sites of AC-stimulated Kv1.2 endocytosis. The physiological significance of secretin mediated suppression of Kv1.2 was supported by collaborative studies which found infusions into the cerebellar cortex of either a toxin that inhibits Kv1.2, or of secretin, enhanced eyeblink conditioning, a form of cerebellar dependent learning, in rats. These studies provided the first evidence that Kv1.2 is regulated by endocytic trafficking in the brain. However, to address the role of that trafficking in synaptic physiology requires knowledge about the determinants of Kv1.2’s endocytic potential, and non-destructive assays to measure Kv1.2 endocytosis in neural circuits. This dissertation therefore concludes with preliminary studies that explore an ancient motif regulating Kv1.2 trafficking, and that discuss a novel dual fluorescent fusion protein reporter of Kv1.2’s subcellular localization.

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