U534918.pdf (11.87 MB)
Molecular properties of recominant large conductance calcium-activated potassium channels
thesisposted on 2014-12-15, 10:34 authored by Jonathan David. Lippiat
1. Currents through large conductance calcium-activated potassium channels were recorded using path clamp from human embryonic kidney (HEK) cells expressing recombinant DNA.;2. The permeation of potassium ions through single cloned rat channels, rSlo+1m was described using Eyring's rate theory. The model included two energy wells and three barriers. N-methyl-D-glucamine was found to block potassium permeation by binding to the intracellular binding site in the pore.;3. A HEK cell line was generated that stably expressed the human bladder BKCa channel subunit (hSlo). Macroscopic currents recorded from patches excised from these cells were activated by depolarisation and by intracellular calcium. The currents also exhibited a voltage- and calcium-dependent inactivation. This was shown to be due to block by contaminant levels of barium and a change in the potassium concentration gradient caused by the large potassium conductance.;4. A phenylalanine residue (F380) in the S6 transmembrane segment was mutated to both isoleucine and tyrosine. Both mutations reduced the unitary conductance and affected the gating of the channel. Mutating F380 to tyrosine enhanced channel opening states whilst mutating to isoleucine inhibited channel opening.;5. Searching EST databases for homologues of the BKCa channel subunit revealed new members. Human 1, 2, 3 and 4 subunits were cloned into bicistronic expression vectors for functional coexpression in HEK cells with the hSlo subunit.;6. The channels comprised of and the different subunits were characterised. The 1, 2 and 4 upregulated the BKCa channel activation kinetics and reduced the sensitivity of the channel to block by iberiotoxin. The 3 subunit had no noticeable effects on either kinetics or toxin block.
Date of award2001-01-01
Author affiliationCell Physiology
Awarding institutionUniversity of Leicester