Abstract
ABSTRACTThe IKs current is important in the heart’s response to sympathetic stimulation. β-adrenergic stimulation increases the amount of IKs and creates a repolarization reserve that shortens the cardiac action potential duration. We have recently shown that 8-CPT-cAMP, a membrane-permeable cAMP analog, changes the channel kinetics and causes it to open more quickly and more often, as well as to higher subconductance levels, which produces an increase in the IKs current. The mechanism proposed to underlie these kinetic changes is increased activation of the voltage sensors. The present study extends our previous work and shows detailed subconductance analysis of the effects of 8-CPT-cAMP on an enhanced gating mutant (S209F) and on a double pseudo-phosphorylated IKs channel (S27D/S92D). 8-CPT-cAMP still produced kinetic changes in S209F + KCNE1, further enhancing gating, while S27D/S92D + KCNE1 showed no significant response to 8-CPT-cAMP, suggesting that these last two mutations fully recapitulate the effect of channel phosphorylation by cAMP.
Highlights
The IKs potassium current is involved in terminating the plateau phase of the cardiac action potential and becomes more important as sympathetic activity increases the heart rate [1]
Two residues in the N-terminus are phosphorylated by cAMP, S27 and S92 [11,12,13] and under sympathetic stimulation, the N-terminus of Q1 becomes phosphorylated by protein kinase A (PKA), which is part of a macromolecular complex bound to the C-terminal domain of Q1 [14]
We showed that the increase in current observed upon 8-CPT-cAMP addition is caused by channels opening more quickly, more often and to higher open sublevels, and suggested that these effects were caused by increased activation of the voltage sensor domains (VSDs) [15]
Summary
The IKs potassium current is involved in terminating the plateau phase of the cardiac action potential and becomes more important as sympathetic activity increases the heart rate [1]. When E1 subunits are a part of the channel complex, the current is converted from one that activates rapidly and inactivates, to one with very slow activation, deactivation kinetics and no inactivation [8,9]. The number of these accessory subunits in the channel complex can vary between 1 to 4 units per channel [10], which allows the channel to be modulated and perhaps regulated by the number of E1s in the complex. Mutant Q1 channels with enhanced or fully activated VS were used to characterize this effect. In this addendum, we have further analysed the single channel kinetics of two Q1 mutants used in the original paper. The other is a double phosphomimetic mutant (S27D/S92D), which appeared to show no response to 8-CPT-cAMP, but that has kinetic properties more similar to wild-type than the single phosphomimetic mutant, S27D
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