Emerging Concepts in Ion Channel Biophysics

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Emerging Concepts in Ion Channel Biophysics Poster Abstracts
48
28-POS Board 28
Effects of Fixed Charge on the Selectivity of Nachbac Channels/Mutants
Olena Fedorenko 1, Carlo Guardiani2, Igor Kaufman 1, Dmitry G. Luchinsky1,3, William A.
Gibby 1, Igor Khovanov2 , Peter V. McClintock1, Stephen K. Roberts 1.
1Lancaster University, Lancaster, United Kingdom, 2University of Warwick, Coventry, United
Kingdom, 3SGT Inc., Greenbelt, MD, USA.
The present study was conducted to apply the ionic Coulomb blockade (ICB) model to bacterial
Nav channels and provide new insights into the role of the fixed charge (Qf) associated with the
selectivity filter (SF). Whole-cell recordings of activity for NaChBac and mutants of different Qf
were analyzed for permeability and selectivity for mono- and di-valent cations. We show that 1)
increasing Qf correlates with a shift in the permeation sequence for monovalent cations to larger
ionic radii (K+, Rb+ and Cs+) and 2) channels with |Qf| ≥ 8 exhibit divalent blockade and
anomalous mole fraction effect in solutions containing mixtures of Na+ and Ca2+; however,
mutants with Qf = -8 generated with different combinations of glutamate and aspartate in the SF
exhibited markedly different cation permeation profiles. These results are interpreted using the
ICB model (extended to include desolvation terms) together with structural insights from
molecular dynamic modelling (MD) of the SF. MD simulations provide an interpretation
consistent with the ICB prediction of a stable state for the neutralized SF and for the increased
Qf mutants requiring a larger number of Na+ than Ca2+ ions to neutralize SF. MD simulations
show that a consequence of higher sodium occupancy in the pore region results in pore closure
and consequently a decrease in the sodium permeation. These predictions are confirmed by
experimental observations. Research is supported by EPSRC (grants No EP/M015831/1 and
EP/M016889/1).