UVM Theses and Dissertations
Format:
Print
Author:
Shortway, Jeffrey H.
Dept./Program:
Pharmacology
Year:
2014
Degree:
M.S.
Abstract:
Kv1 family potassium channels are the predominant contributor to delayed rectifier currents in cerebral arteriole vascular smooth muscle cells, and are therefore likely to be key determinants of cerebral blood flow. They are also widely expressed in the nervous system. Kv1 channels consist of four membrane spanning alpha-subunit proteins that can assemble into homomultimeric channels consisting of four identical subunit isotypes, or heteromultimeric channels consisting of up to four different alphasubunit isotypes. Heteromultimeric channels often combine the electrophysiological properties of homomultimeric channels composed of its constituent alpha-subunits, but less is known how heteromultimerization affects channel regulation.
Kv1.2 and Kv1.5 are 2 members of the Shaker-related family of voltage-sensitive potassium channels and contribute to regulation of membrane excitability. Homomultimeric Kv1.2 and homomultimeric Kvl. 5 channels are regulated by tyrosine phosphorylation and subsequent endocytosis of the channel from the plasma membrane. However, Kv1.2/Kv1.5 heteromultimeric channels have key roles in the vasculature and nervous system, but little is known about how these heteromultimeric channels are regulated. Using flow cytometry, we compared the mechanisms controlling endocytic trafficking of Kv1.2 homomultimeric vs. Kv1.2/Kv1.5 heteromultimeric channels expressed in HEK293 cells.
The Kv1.2 homomultimeric channel is regulated by endocytosis through a dynamin dependent pathway. We found that, in contrast, the Kv1.2/Kv1.5 heteromultimeric channel was insensitive to pharmacological inhibition of dynamin. Endocytosis of Kv1.2 also requires activity of the small GTPase RhoA. We found that pharmacological inhibitors of RhoA affected Kv1.2 homomultimeric but not Kv1.2/Kv1.5 heteromultimeric channels. This indicates that Kv1.2/1.5 heteromultimers are uncoupled from the RhoA pathways. The heteromultimer was nevertheless sensitive to protein kinase C or Src-kinase dependent endocytosis, demonstrating that it retains its ability to be regulated by pathways that do not involve RhoA.
Kv1.2 and Kv1.5 are 2 members of the Shaker-related family of voltage-sensitive potassium channels and contribute to regulation of membrane excitability. Homomultimeric Kv1.2 and homomultimeric Kvl. 5 channels are regulated by tyrosine phosphorylation and subsequent endocytosis of the channel from the plasma membrane. However, Kv1.2/Kv1.5 heteromultimeric channels have key roles in the vasculature and nervous system, but little is known about how these heteromultimeric channels are regulated. Using flow cytometry, we compared the mechanisms controlling endocytic trafficking of Kv1.2 homomultimeric vs. Kv1.2/Kv1.5 heteromultimeric channels expressed in HEK293 cells.
The Kv1.2 homomultimeric channel is regulated by endocytosis through a dynamin dependent pathway. We found that, in contrast, the Kv1.2/Kv1.5 heteromultimeric channel was insensitive to pharmacological inhibition of dynamin. Endocytosis of Kv1.2 also requires activity of the small GTPase RhoA. We found that pharmacological inhibitors of RhoA affected Kv1.2 homomultimeric but not Kv1.2/Kv1.5 heteromultimeric channels. This indicates that Kv1.2/1.5 heteromultimers are uncoupled from the RhoA pathways. The heteromultimer was nevertheless sensitive to protein kinase C or Src-kinase dependent endocytosis, demonstrating that it retains its ability to be regulated by pathways that do not involve RhoA.