Section I{KS}, the slowly activating delayed rectifier potassium (K[+]) current in the heart is critical importance to human physiology as evident from the fact that mutations in either its ? (KCNQ1) or ? (KCNE1) subunit have been linked to multiple cardiac arrhythmia syndromes, including long QT syndrome (LQTS); short QT syndrome; and familial atrial fibrillation. The I{KS} channel is upregulated during sympathetic stimulation by PKA phosphorylation, which contributes critically to the physiological shortening of cardiac action potentials in response to sympathetic nerve activity. This shortening is necessary to ensure adequate ventricular filling time with accompanying increases in heart rate. It is also during sympathetic stimulation that most sudden deaths from LQTS occur. Understanding the mechanisms that underlie these mutation-induced arrhythmia syndromes requires unraveling the molecular interactions between KCNQ1 and KCNE1 within the context of normal and disease altered I{KS} channels. But to date, the critical questions of how KCNE1 alters KCNQ1 channel gating and how I{KS} channels are modulated by PKA are still not fully answered. Our previous work has revealed that Beta-AR regulation of channels requires assembly of a macromolecular complex that includes both KCNQ1 and KCNE1, as well as the adaptor protein Yotiao (AKAP 9). We will here use novel nanobodies to deliver regulatory domains of PKA directly to the KCNQ1/KCNE1 channel with and without co-assembly with AKAP9. These experiments will allow dissection of the critical role of AKAP9 in the delivery of signaling molecules to KCNQ1/KCNE1 from additional putative modulatory roles of the AKAP in modulating channel function post phosphorylation. In the recent CryoEM structure of KCNQ1 putative interacting residues between KCNQ1 and KCNE1 map between the VSD and PD, suggesting that KCNE1 is located in this area of the KCNQ1 structure. We will test whether KCNQ1 and KCNQ1/KCNE1 channels open using different gating hinges in S6. We will here also identify KCNQ1-KCNE1 interacting residues and determine whether these residues affect the different gating hinges. PKA has been shown to alter the voltage dependence, sub-conductance occupancy, and kinetics of I{KS} channels. Using voltage clamp fluorometry together with mutations and PIP2 depletion that uncouple the VSD and PD, we will determine whether PKA affect the VSD, PD, and/or VSD-to-PD coupling in I{KS} channels. The anticipated results of these experiments will provide a structural basis for control by PKA and KCNE1 of the physiological function of this critical ion channel and will also provide novel targets for the development of drugs to modulate its activity. This would be a milestone toward mutation-specific treatments of diseases, such as cardiac arrhythmias, caused by mutations in KCNQ1 and KCNE1.

Public Health Relevance

Section Mutations and variants in the IKS potassium channel ? (KCNQ1) and ? (KCNE1) subunits as well as the adaptor protein AKAP9 which coordinates the IKS macromolecular signaling complex have been implicated in a number of life threatening cardiac arrhythmias. Here we will here determine how the IKS channel is regulated by PKA and how assembly with different numbers of its ancillary subunits contributes to normal and pathological function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM109762-07
Application #
10079488
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Nie, Zhongzhen
Project Start
2014-05-01
Project End
2022-12-31
Budget Start
2021-01-01
Budget End
2021-12-31
Support Year
7
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Miami School of Medicine
Department
Physiology
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
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