Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Poster Abstracts

64-POS Board 64 Gating Modifier Toxins as Hits for Developing Blockers of Nav1.4 Sodium Channel Omega Currents: Domain I-Specific Effect of Spider Toxin Hm-3 Roope Männikkö 1 , Zakhar O. Shenkarev 2,3 , Michael G. Thor 1 , Antonina A. Berkut 2,3 , Mikhail Yu. Myshkin 2,3 , Alexander S. Paramonov 2 , Dmitry S. Kulbatski 2,4 , Kuzmin Dmitry 5 , Marisol Sampedro Castañeda 1 , Louise King 1 , Emma R. Wilson 1 , Ekaterina N. Lyukmanova 2,4 , Mikhail P. Kirpichnikov 2,4 , Stephanie Schorge 1,5 , Frank Bosmans 6 , Michael G. Hanna 1 , Dimitri Kullmann 1,5 , Alexander A. Vassilevski 2 . 1 MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology, London, United Kingdom, 2 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation, 3 Moscow Institute of Physics and Technology (State University), Moscow, Russian Federation, 4 Biological Faculty, Lomonosov Moscow State University, Moscow, Russian Federation, 5 Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, United Kingdom, 6 Johns Hopkins University School of Medicine, Dept of Physiology and S H Snyder Dept of Neuroscience, Baltimore, MD, USA. Hypokalaemic periodic paralyses (HypoPP) are rare channelopathies characterized by episodes of severe muscle weakness associated with low serum potassium levels, often progressing to permanent muscle weakness with age. Depolarizing gating pore currents caused by mutations in arginine residues in the voltage-sensing domains (VSDs) of skeletal muscle voltage-gated sodium and calcium channels are the underlying molecular pathomechanism. There is currently no effective, mechanistically-based treatment for HypoPP. We present the functional characterization of an Na v 1.4 channel mutant previously identified in a HypoPP patient, p.R222W. The mutation neutralizes the second S4 arginine in domain I of the channel causing gating pore currents. We tested if these currents can be inhibited by gating modifier toxin Hm-3, derived from the venom of the crab spider Heriaeus melloteei . The toxin inhibits gating pore currents (IC 50 =5.4µM) from channels with mutations in the second arginine in VSD-I (p.R222W and p.R222G), but not from channels with analogous mutations in VSD-II or -III. Mutations in VSD-I reduced Hm-3 gating modifier effect, confirming the domain specificity of Hm-3. NMR studies of the VSD-I of Na v 1.4 with Hm-3 demonstrate electrostatic and hydrophobic interactions of the toxin with the S3b helix and S3-S4 extracellular loop. Implanting the S3-S4 helix-loop-helix of VSD-I, but not VSD-II, -III or -IV, to the corresponding location of K v 2.1 conveyed Hm-3 sensitivity to the host channel. Our data identify a novel and specific binding site for neurotoxins on the S3-S4 linker region of the VSD-I of Na v 1.4 and highlight gating modifier toxins as useful hits in the development of omega current blockers for HypoPP therapy.

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