Structures of ion channel complexes

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Program Long-term Objective: To better understand biophysical mechanisms of normal gating in potassium Kv7.1 (KCNQ1 or Q1) ion (K+) channels, complexed with calmodulin and KCNE subunits, E(x). We described the flexible stoichiometry, a novel inactivation mechanism, the allosteric gating of the Q1:E1 channel complex, and adopted state-of-the art cryo-EM technology during the prior granting period. Now, we will combine cryo-EM with cellular biophysical methods such as whole-cell and single channel patch recording, single oocyte fluorescence spectroscopy, unnatural amino acid crosslinking, and molecular dynamics (MD) simulation, to address four short-term objectives: 1) Molecular binding of inhibitors to rapidly activating Kv7.1 channels (Q1): Understanding mechanisms of action of inhibitors such as HMR1556, UCL2077, isofluranes, and their direct or allosteric binding will reveal how Q1 complexes operate and are inhibited. Ability to obtain high resolution cryo-EM structures of Q1, and eventually Q1:E(x) allows us to test structural and biophysical properties of different inhibitors complexed with various channel stoichiometries. 2) Cryo-EM and biophysics of activation gating in stoichiometrically fixed complexes of Q1+ E(x) channels: We will develop our existing Q1 cryo-EM methodology to resolve the first human Q1:E(1) structure. As Q1:E1 channels form the most important complexes of KCNQ1 and KCNE(x) (no structures obtained to date), such a structure would have wide scientific utility in the design and bioengineering of compounds that modulate channel complex activity. 3) Elucidation of resting-closed state Kv7.1 and Q1:E1 channel structures: Two vital structural elements subserve the function of voltage-activated K+ channels: the voltage sensor (VS) and the pore domain, but currently there are no structural representations of resting closed K+ channels with resting VS. We will use the E160R and/or E160R/R228E mutations to restrain channels in their resting states and obtain cryo-EM resolution of closed-state Q1:E(x) channel structures. 4) Markov and MD simulations of channel dynamics, drug binding/unbinding: Structural representations of the channel complexes will guide further cryo-EM studies and constrain Markov modeling and MD simulations of channel dynamics, to allow us to construct dynamic models of channel activation and inhibition. HQP: Our welcoming, rigorous, and diverse learning environment will help HQP acquire necessary skills to achieve independent and rewarding professional careers. Impact: Cryo-EM and biophysical studies of Q1 and Q1:E(x) inhibition and gating will uncover molecular events underlying function of these biological gatekeepers, and improve understanding of cell electrical activity. Knowledge will be disseminated via publication, invited conference, and this work is anticipated to lead to further NSERC-partnered opportunities, e.g., IRAP, Alliance, to generate further scientific, training, and economic opportunities.