The structure of a voltage gated potassium channel

Voltage gated potassium channels (Kv channels) are tetrameric ion channels, responsible for regulating the potassium component of the membrane potential in a large range of cell types ranging from mammalian excitable cells to bacteria. Attempts have been made to elucidate the structure of voltage gated potassium channels using X-ray crystallography, however due to the inherent flexibility of the voltage sensors, removal of the channels from their native lipid environment causes distortion of the channels, and as a result much controversy remains over their exact structure. KvAP is a voltage gated potassium channel from the thermophilic archaea Aeropyrum pernix which contains a single cysteine residue, which can be removed by site directed mutagenesis to give a template for cysteine scanning mutagenesis. Fluorescence spectroscopy utilising cysteine reactive probes can be used to probe the membrane topology of proteins in the context of a lipid bilayer. Single cysteine mutants within the pore domain outer helix (S5 helix) of KvAP were generated and labelled with thiol reactive fluorescent probes. These probes were used to report on the polarity of the surrounding environment using a combination of the environmental sensitivity of the probes and fluorescence quenching from both aqueous and lipid phases. Fluorescence results fit well to a hypothetical model describing a trough like variation in dielectric constant of the membrane, allowing the determination of the position of the hydrophobic interface of the membrane at each end of the helix. A mutant of KvAP with no voltage sensing domains was also generated and subjected to cysteine scanning mutagenesis of the S5 helix. Again results fitted well to a hypothetical profile of the dielectric constant of the membrane, and the shift in fluorescence properties at some positions within the helix in the absence of the voltage sensor shows the residues of the pore domain which are in close contact with the voltage sensor.

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Southampton

Year dc:date.issued

2010

Author dc:creator

• Rogers, Nik

Advisors dc:contributor.advisor

• Lee, A.G.
• East, J. Malcolm