Structure and function of potassium and calcium channels
In this thesis, potassium channels human Kv2.1 and rat Kv2.1, along with calcium channels Ca, 1.2, Ca, 3.1, and chimeras have been studied. These channels were expressed in Xenopuso ocytesf or electrophysiological experiments using two electrode voltage clamp. For protein expression studies,DNA was expressed in BL21 or COS-7 cells and purified using glutathione or an ID4 affinity column. Firstly, the roles of the N- and C- terminal domains in the activation kinetics of rat and human forms of Kv2.1 were investigated. A mutant in the N- terminal domain and chimeras between the rat and human forms were constructed. All clones were expressed in Xenopuso ocytesa nd activation times obtained. The results suggested that key residues in the N- and C- terminal domains are involved in determining the activation kinetics of rat and human Kv2.1. Further experiments were carried out using GST fusion proteins, Biacore surface plasma resonance and FRET investigations. These confirmed that the N and C- terminal domains are important in determining activation kinetics, and that these regions interact. To determine the positions of both the N- and C- terminal domains in the folded channel, rat Kv2.1 and a C-terminal deleted protein were expressed purified, and shown to have correct protein folding. These samples were sent for electron microscopy experiments and a preliminary picture obtained. No studies of S4 movement in calcium channels have been reported previously. Cysteine residues were substituted into the domain I S4 of a calcium channel chimera (with domain I of Ca, 3.1 replaced by Caj. 2). Cysteine residues at positions 263,265,266,268,269 and 271 were characterized by electrophysiology. Cysteine mutants at residues 263,265,266 and 268 reacted when extracellular PCMBS was applied, but mutants at residues 269 and 271 did not. This suggests that under depolarising conditions the S4 segment is exposed to the extracellular environment up to and including residue 268, with residues 269 and 271 remaining buried. Further investigation of residue 263 indicated that this movement occurs at potentials more negative than the resting membrane potential of -80mV. The data suggests that under depolarisation, the S4 becomes exposed to the extracellular solution and this movement of the S4 occurs before the ionic flow.