Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Poster Abstracts

28-POS Board 28 Effects of Fixed Charge on the Selectivity of Nachbac Channels/Mutants

Olena Fedorenko 1 , Carlo Guardiani 2 , Igor Kaufman 1 , Dmitry G. Luchinsky 1,3 , William A. Gibby 1 , Igor Khovanov 2 , Peter V. McClintock 1 , Stephen K. Roberts 1 . 1 Lancaster University, Lancaster, United Kingdom, 2 University of Warwick, Coventry, United Kingdom, 3 SGT 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).

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