U536424.pdf (6.91 MB)
Studies of voltage-activated potassium channels in a mammalian cell line
thesisposted on 2014-12-15, 10:35 authored by Iftikhar Ahmad. Khan
The biophysical properties of delayed rectifier (Kv1.5) and an A-type (Kv4.2) K+-channel were investigating using a Murine Erythroleukemia (MEL) cell line. Genomic DNA clones of full length human Kv1.5 gene (isolated and sequenced from an adult heart cDNA library) and a cDNA sequence encoding Kv4.2 (isolated from rat hippocampus) were stably transfected into MELL cells by electroporation. Gene expression was regulated by the locus control region of the human -globin gene; channels were expressed after induction of cells to undergo erythroid differentiation by addition of dimethyl sulphoxide.;The whole cell configuration of the patch clamp technique was used to characterise the biophysical properties of the two channels, which are distinguishable by rates of current inactivation. The voltage-dependence of current activation was used to construct current-voltage relationships. Selectivity of Kv1.5 for K+ ions was assessed, and reversal potentials measured with different external K+ concentrations. The voltage-dependence of steady state activation, (a measure of the channel open probability) and steady state inactivation were examined, and the time course of activation and inactivation calculated in terms of classical Hodgkin-Huxley equations.;Synthetic 'ball' peptides based upon the sequence of the amino-terminal structural motif that causes fast inactivation (analogous to a 'ball and chain' occluding the pore internally) in Shaker K+ channels were included in pipette solutions during recordings. Blocking/unblocking rates, and association/dissociation rate constants were calculated to characterise the effect on Kv1.5 and Kv4.2 currents. Kv1.5 does not undergo fast inactivation unlike Kv4.2, but these peptides caused Kv1.5 inactivation to speed up to resemble an A-type current. The mutation L7E caused peptides to become inactive; the double mutation E12K,D13K made the peptide more potent. Even Kv4.2 currents inactivated significantly more quickly. These results imply that there is a high degree of conservation of the ball receptor from Shaker to Kv4.2 and Kv1.5.
Date of award1998-01-01
Author affiliationCell Physiology and Pharmacology
Awarding institutionUniversity of Leicester