Project Details
Description
For year nine of the grant Dr. Vanoye will be at Northwestern University as of April 1st, 2014. During this time he will design and perform electrophysiological and biochemical experiment to address specific questions formulated to further develop our KCNQ1-KCNE1 and KCNQ1-KCNE3 complex models in order to understand how these KCNE proteins modulate KCNQ1 channel activity.
Aim 2: mutagenesis of KCNE1 and KCNQ1, biochemical analysis, and electrophysiological recordings of the KCNQ1-KCNE1 channel complex to evaluate and refine our current working model for how KCNE1 alters KCNQ1 channel gating and open state conductance.
Our structural KCNQ1-KCNE1 channel complex model predicts interactions between the KCNQ1 S4-S5 linker and the intracellular region of the KCNE1 transmembrane domain. We will first focus on residues V241 and Q244 and their interactions with KCNE1 residue Ser68 and selected adjacent sites (based on results for Ser68Cys). Associations/interactions between KCNQ1 and KCNE1 residues will be tested by substitution with cysteine and testing for cys-cys bond formation by intracellular perfusion with redox agents copper phenanthroline and DTT.
Aim 4: structure-function and biochemical analysis of the working model for how KCNE3 rapidly activates KCNQ1 channel function.
In this phase, interaction sites between KCNQ1 and KCNE3 that are predicted by our recent KCNQ1-KCNE3 complex models will be tested. First we will focus the predicted interactions between KCNQ1 residues V241 and/or Q244 with KCNE3 residue S82. Interestingly, these KCNQ1 residues are also predicted to be critical for the KCNQ1-KCNE1 complex. These observations are in agreement with our “compare and contrast” analytical approach to develop a structural understanding of both the similarities and differences in how KCNE1 and KCNE3 modulate KCNQ1.
Associations/interactions between KCNQ1 and KCNE3 residues will be tested by substitution with cysteine and testing for cys-cys bond formation by intracellular perfusion with redox agents copper phenanthroline or DTT. Once this interaction is confirmed, we will use this experimentally-defined constraint to predict more interaction sites between these proteins and then test experimentally.
These results will allow us to refine our KCNQ1-KCNE channel complex models and develop stronger structural models for how KCNE proteins modulate KCNQ1 activity in health and disease.
Status | Finished |
---|---|
Effective start/end date | 4/1/14 → 3/31/16 |
Funding
- Vanderbilt University (VUMC42969//5R01DC007416-09)
- National Institute on Deafness and Other Communication Disorders (VUMC42969//5R01DC007416-09)
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