The long-range goal of this project is to further our understanding of how K+ channels work at a molecular level. The project focuses on the interaction between a Shaker K+ channel from Drosophila melanogaster, and charybdotoxin (CTX), a pore-blocking peptide inhibitor. CTX will be used to probe the external entryway to the conduction pore of this genetically manipulable channel, in order to identify and characterize this important part of the ion channel. Shaker K+ channels will be expressed in Xenopus oocytes and studied using electrophysiological techniques. CTX-binding regions will first be identified by producing site-directed mutant Shaker K+ channels that display altered CTX inhibition. A major effort will be put towards understanding the molecular mechanism by which a given mutation influences the interactions with toxin. For example, a particular glutamate on the channel has already been shown to influence the binding of the cationic CTX molecule by a simple through-space electrostatic mechanism; the glutamate must therefore be physically close to the CTX binding site. The next step in the project will be to ask if the amino acid residues which affect toxin binding also play a role in the ion conduction process. This will be determined by studying the single-channel current properties of the mutant Shaker K+ channels. The expectation is, based on preliminary results, that CTX will point out anionic residues in the channel's outer mouth which influence conducting ions by an electrostatic mechanism. This project will help to define the transmembrane orientation of the Shaker K+ channel (because the toxin blocks only from the outside), it will identify residues that make up the channels's external conduction pore entryway, and it may point out adjacent protein domains that form the deeper regions of the pore. The proposed project is health related; many cellular processes that are operating in virtually every organ system of the human body depend on K+ channels.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043949-02
Application #
3303075
Study Section
Physiology Study Section (PHY)
Project Start
1990-04-01
Project End
1995-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
2
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Lee, Chia-Hsueh; MacKinnon, Roderick (2018) Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures. Science 360:508-513
Wang, Weiwei; MacKinnon, Roderick (2017) Cryo-EM Structure of the Open Human Ether-à-go-go-Related K+ Channel hERG. Cell 169:422-430.e10
Lee, Chia-Hsueh; MacKinnon, Roderick (2017) Structures of the Human HCN1 Hyperpolarization-Activated Channel. Cell 168:111-120.e11
Tao, Xiao; Hite, Richard K; MacKinnon, Roderick (2017) Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel. Nature 541:46-51
Hite, Richard K; Tao, Xiao; MacKinnon, Roderick (2017) Structural basis for gating the high-conductance Ca2+-activated K+ channel. Nature 541:52-57
Hite, Richard K; MacKinnon, Roderick (2017) Structural Titration of Slo2.2, a Na+-Dependent K+ Channel. Cell 168:390-399.e11
Whicher, Jonathan R; MacKinnon, Roderick (2016) Structure of the voltage-gated K? channel Eag1 reveals an alternative voltage sensing mechanism. Science 353:664-9
Su, Zhenwei; Brown, Emily C; Wang, Weiwei et al. (2016) Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels. Proc Natl Acad Sci U S A 113:5748-53
Touhara, Kouki K; Wang, Weiwei; MacKinnon, Roderick (2016) The GIRK1 subunit potentiates G protein activation of cardiac GIRK1/4 hetero-tetramers. Elife 5:
Wang, Weiwei; Touhara, Kouki K; Weir, Keiko et al. (2016) Cooperative regulation by G proteins and Na(+) of neuronal GIRK2 K(+) channels. Elife 5:

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