Role of the cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) domain in trafficking and stabilization of Kv11.1 channels.
The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.
From the Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, 405 Liverpool Street and St. Vincent's Clinical School, University of New South Wales, Victoria Street, Darlinghurst, New South Wales 2010, Australia.
Ying
Y
Ke
From the Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, 405 Liverpool Street and.
Mark J
MJ
Hunter
From the Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, 405 Liverpool Street and St. Vincent's Clinical School, University of New South Wales, Victoria Street, Darlinghurst, New South Wales 2010, Australia.
Chai Ann
CA
Ng
From the Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, 405 Liverpool Street and St. Vincent's Clinical School, University of New South Wales, Victoria Street, Darlinghurst, New South Wales 2010, Australia.
Matthew D
MD
Perry
From the Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Victor Chang Cardiac Research Institute, 405 Liverpool Street and St. Vincent's Clinical School, University of New South Wales, Victoria Street, Darlinghurst, New South Wales 2010, Australia j.vandenberg@victorchang.edu.au.
Jamie I
JI
Vandenberg
D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport
D051638:Ether-A-Go-Go Potassium Channels
PAS Domain; Potassium Channels; Site-directed Mutagenesis; Subunit-Subunit Interaction; Trafficking; Western Blotting; hERG