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Regulation of the IKs current by PKA phosphorylation Thompson, Emely Rose McKinley

Abstract

The IKs current, which is composed of KCNQ1 and KCNE1 subunits, increases in size as a result of beta-adrenergic stimulation. This response is a result of the KCNQ1 subunit being phosphorylated by protein kinase A (PKA). This is an important physiological response at high heart rates that allows the ventricles adequate time to fill. Mutations in either of these subunits can cause impaired cardiac repolarization and result in long and short QT syndromes, as well as familial atrial fibrillation. The mechanism by which the channel complex reacts to this stimulation has not been fully elucidated. To investigate the mechanism behind this, both total internal reflection florescence microscopy (TIRF) and single-channel recording were used. 8-CPT-cAMP, a membrane-permeant analog of cAMP, was used to induce PKA phosphorylation of KCNQ1. TIRF studies found no significant change in the number of channels at the cell surface. Single-channel recordings of IKs had a reduced first latency to opening showing that the channel opened more quickly in response to 8-CPT-cAMP. The IKs current has multiple open states and, in the presence of 8-CPT-cAMP, occupied the higher subconducting states more frequently. An increase in the open probability of the channel complex was also seen. In response to phosphorylation triggered by 8-CPT-cAMP, the first latency to opening of the channel is reduced and the channel opens more quickly, more often and passes more current by increasing the higher conducting open states. This results in an increase in IKs current at the macroscopic level. Using enhanced gating mutant KCNQ1 channels, it is shown that the effect of phosphorylation is likely through further activation of the voltage sensor. KCNE1 is required for a functional response to PKA phosphorylation. Both whole-cell and single-channel recordings show that as the number of KCNE1 subunits is reduced, a graded effect is seen in response to 8-CPT-cAMP; the less KCNE1 subunits, the smaller the response. However, in single-channel recordings, there was also a KCNE1-independent effect of phosphorylation as the first latencies for all KCNQ1-KCNE1 complexes were reduced in a non-graded manner. This suggests that phosphorylation may have both KCNE1-dependent and independent effects on the channel.

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