Photoaffinity Ligand for the Inhalational Anesthetic Sevoflurane Allows Mechanistic Insight into Potassium Channel Modulation.
Sevoflurane is a commonly used inhaled general anesthetic. Despite this, its mechanism of action remains largely elusive. Compared to other anesthetics, sevoflurane exhibits distinct functional activity. In particular, sevoflurane is a positive modulator of voltage-gated Shaker-related potassium channels (Kv1.x), which are key regulators of action potentials. Here, we report the synthesis and validation of azisevoflurane, a photoaffinity ligand for the direct identification of sevoflurane binding sites in the Kv1.2 channel. Azisevoflurane retains major sevoflurane protein binding interactions and pharmacological properties within in vivo models. Photoactivation of azisevoflurane induces adduction to amino acid residues that accurately reported sevoflurane protein binding sites in model proteins. Pharmacologically relevant concentrations of azisevoflurane analogously potentiated wild-type Kv1.2 and the established mutant Kv1.2 G329T. In wild-type Kv1.2 channels, azisevoflurane photolabeled Leu317 within the internal S4-S5 linker, a vital helix that couples the voltage sensor to the pore region. A residue lining the same binding cavity was photolabeled by azisevoflurane and protected by sevoflurane in the Kv1.2 G329T. Mutagenesis of Leu317 in WT Kv1.2 abolished sevoflurane voltage-dependent positive modulation. Azisevoflurane additionally photolabeled a second distinct site at Thr384 near the external selectivity filter in the Kv1.2 G329T mutant. The identified sevoflurane binding sites are located in critical regions involved in gating of Kvchannels and related ion channels. Azisevoflurane has thus emerged as a new tool to discover inhaled anesthetic targets and binding sites and investigate contributions of these targets to general anesthesia.
Department of Anesthesiology & Critical Care, University of Pennsylvania Perelman School of Medicine , 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104, United States.|Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine , 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104, United States.
Kellie A
KA
Woll
Department of Chemistry, University of Pennsylvania School of Arts and Sciences , 231 S. 34th Street, Philadelphia, Pennsylvania 19104, United States.
Wesley
W
Peng
Department of Neuroscience and Vickie and Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College, Thomas Jefferson University , 900 Walnut Street, JHN 417, Philadelphia, Pennsylvania 19107, United States.
Qiansheng
Q
Liang
Department of Neuroscience and Vickie and Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College, Thomas Jefferson University , 900 Walnut Street, JHN 417, Philadelphia, Pennsylvania 19107, United States.
Lianteng
L
Zhi
Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine , 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104, United States.
Jack A
JA
Jacobs
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine , Philadelphia, Pennsylvania 19129, United States.
Lina
L
Maciunas
Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania , 3400 Civic Center, Building 421, Philadelphia, Pennsylvania 19104, United States.
Natarajan
N
Bhanu
Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania , 3400 Civic Center, Building 421, Philadelphia, Pennsylvania 19104, United States.
Benjamin A
BA
Garcia
Department of Neuroscience and Vickie and Jack Farber Institute for Neuroscience, Sidney Kimmel Medical College, Thomas Jefferson University , 900 Walnut Street, JHN 417, Philadelphia, Pennsylvania 19107, United States.
Manuel
M
Covarrubias
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine , Philadelphia, Pennsylvania 19129, United States.
Patrick J
PJ
Loll
Department of Chemistry, University of Pennsylvania School of Arts and Sciences , 231 S. 34th Street, Philadelphia, Pennsylvania 19104, United States.
William P
WP
Dailey
Department of Anesthesiology & Critical Care, University of Pennsylvania Perelman School of Medicine , 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104, United States.
Roderic G
RG
Eckenhoff
D018685:Anesthetics, Inhalation; D000818:Animals; D001665:Binding Sites; D006801:Humans; D015640:Ion Channel Gating; D051663:Kv1.2 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008738:Methyl Ethers / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D019920:Photoaffinity Labels; D000077149:Sevoflurane; D051637:Shaker Superfamily of Potassium Channels; D014982:Xenopus laevis
D018685:Anesthetics, Inhalation; D051663:Kv1.2 Potassium Channel; D008024:Ligands; D008738:Methyl Ethers; D019920:Photoaffinity Labels; D051637:Shaker Superfamily of Potassium Channels; D000077149:Sevoflurane