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Emerging Concepts in Ion Channel Biophysics
Poster Abstracts
60
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.