Probing the mechanisms underlying modulation of quinidine sensitivity to cardiac I(Ks) block by protein kinase A-mediated I(Ks) phosphorylation.
OBJECTIVE
Cardiac I(Ks) is enhanced by protein kinase A (PKA) stimulation. And PKA-stimulated I(Ks) is about threefold less sensitive to quinidine block than basal current. In this study, we further tested two competing hypotheses: I(Ks) phosphorylation either (i) modulates access of blocking drugs to a binding site; or (ii) destabilizes the drug-channel interaction.


METHODS
To distinguish between these hypotheses, we studied quinidine block of I(Ks) channels in which three PKA site residues of the alpha-subunit KCNQ1 were mutated with a bulky negative charged aspartic acid (D). To study alleviation of I(Ks) block by quinidine, we compared activating current at +60 mV, either with or without 5 s hyperpolarizing prepulses to -120 mV.


RESULTS
Without PKA stimulation, quinidine (100 microM) blocked wild-type current to a similar extent with and without the prepulse (93 +/- 2% of pre-drug current at +60 mV vs. 95 +/- 1%). With PKA-stimulated wild-type channels, however, there was less block with the hyperpolarization to -120 mV: at +60 mV, block was 71 +/- 2% (-prepulse) versus 58 +/- 3% (+prepulse). Individual D-mutations and the triple-D mutant were resistant to quinidine block similar to that seen with PKA-stimulated wild-type I(Ks).


CONCLUSIONS
We conclude that phosphorylation-induced insertion of bulky negative charges alleviates quinidine block and that PKA-induced stimulation, by conferring negative charges to the channels, blunts I(Ks) block as the interaction between the channels and blockers becomes destabilized. These effects would be of clinical significance in providing protective mechanisms against pro-arrhythmias caused by drug-induced inhibition of I(Ks) and I(Kr).
Yang
Tao
T
Oates Institute for Experimental Therapeutics, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-0575, USA. tao.yang@vanderbilt.edu
Kanki
Hideaki
H
Zhang
Wei
W
Roden
Dan M
DM
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024642:Potassium Channels, Voltage-Gated / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011802:Quinidine / Q000378:metabolism*
C489622:KCNE1 protein, human; D051657:KCNQ1 Potassium Channel; D026902:Potassium Channel Blockers; D024642:Potassium Channels, Voltage-Gated; D017868:Cyclic AMP-Dependent Protein Kinases; D011802:Quinidine