UVM Theses and Dissertations
Format:
Print
Author:
Nesti, Edmund Darey
Dept./Program:
Pharmacology
Year:
2006
Degree:
PhD
Abstract:
Voltage-gated ion channels reside in cell membranes and are responsible for generating electrical signals that are vital to cell function. Kv1.2 is a voltage-gated potassium channel expressed in a variety of tissues including the nervous system where it affects neuronal integration and synaptic transmission and, the cardiovascular system where it affects blood flow. Regulation of Kv1.2 function is therefore a means to modulate these systems.
The G-protein coupled MI muscarinic acetylcholine receptor and the epidermal growth factor receptor tyrosine kinase both growth factor receptors trigger the direct tyrosine phosphorylation of Kv1.2. That phosphorylation in turn evokes a strong suppression of Kv1.2 ionic current. This suppression requires activation of the cytoskeleton remodeling protein RhoA and the actin binding protein cortactin, both of which interact directly with Kv1.2, linking it to actin dynamics. Because actin remodeling by cortactin is known to contribute to membrane protein endocytosis we hypothesized that tyrosine phosphorylation dependent suppression of Kv1.2 ionic current involves the tyrosine kinase and actin dependent trafficking of the channel protein from the cell surface through endocytosis.
We have found that the same stimuli that evoke suppression of the Kv1.2 ionic current also cause Kv1.2. to undergo endocytosis. The process is tyrosine phosphorylation-dependent because the same point mutation of Kv1.2 that confers resistance to channel suppression (Y132F) also confers resistance to channel endocytosis. Inhibiting endocytosis with the over expression of a dominant negative form of dynamin blocked stimulus-induced Kv1.2 endocytosis and also blocked suppression of Kv1.2 ionic current. These findings indicate that endocytosis of Kv1.2 from the cell surface is a key mechanism for channel suppression.
Recently, modification of receptor tyrosine kinases by ubiquitin has been identified as an important step leading to endocytosis. Here, we demonstrate that regulation of Kv1.2 includes region specific mono-ubiquitination of the channel. However, mutagenesis within the N- and C-termini of Kv1.2 reveal that ubiquitination of each terminus has a distinct regulatory effect on the channel. We also show that within the C-terminus, the beta subunit of the channel acts as a switch governing the polarity of channel trafficking in response to ubiquitination. We propose that mono-ubiquitination allows both positive and negative regulation of Kv1.2 depending on the site of its ubiquitination and the presence or absence of the Kv1.2 beta subunit.
The G-protein coupled MI muscarinic acetylcholine receptor and the epidermal growth factor receptor tyrosine kinase both growth factor receptors trigger the direct tyrosine phosphorylation of Kv1.2. That phosphorylation in turn evokes a strong suppression of Kv1.2 ionic current. This suppression requires activation of the cytoskeleton remodeling protein RhoA and the actin binding protein cortactin, both of which interact directly with Kv1.2, linking it to actin dynamics. Because actin remodeling by cortactin is known to contribute to membrane protein endocytosis we hypothesized that tyrosine phosphorylation dependent suppression of Kv1.2 ionic current involves the tyrosine kinase and actin dependent trafficking of the channel protein from the cell surface through endocytosis.
We have found that the same stimuli that evoke suppression of the Kv1.2 ionic current also cause Kv1.2. to undergo endocytosis. The process is tyrosine phosphorylation-dependent because the same point mutation of Kv1.2 that confers resistance to channel suppression (Y132F) also confers resistance to channel endocytosis. Inhibiting endocytosis with the over expression of a dominant negative form of dynamin blocked stimulus-induced Kv1.2 endocytosis and also blocked suppression of Kv1.2 ionic current. These findings indicate that endocytosis of Kv1.2 from the cell surface is a key mechanism for channel suppression.
Recently, modification of receptor tyrosine kinases by ubiquitin has been identified as an important step leading to endocytosis. Here, we demonstrate that regulation of Kv1.2 includes region specific mono-ubiquitination of the channel. However, mutagenesis within the N- and C-termini of Kv1.2 reveal that ubiquitination of each terminus has a distinct regulatory effect on the channel. We also show that within the C-terminus, the beta subunit of the channel acts as a switch governing the polarity of channel trafficking in response to ubiquitination. We propose that mono-ubiquitination allows both positive and negative regulation of Kv1.2 depending on the site of its ubiquitination and the presence or absence of the Kv1.2 beta subunit.