Fig. 4: Calcium induced ion-channel properties of reconstituted δ-LIT variants within lipid bilayers.
From: Molecular architecture of black widow spider neurotoxins
Current–Voltage ramp recordings at different cis/trans buffer conditions. a Precursor δ-LIT shows in asymmetric KCl buffer a noisy linear current–voltage relation with reversal potential of Vrev = +4 mV, indicating a slight cation selective channel (\({P}_{{{{{{{\rm{K}}}}}}}^{+}}/{P}_{{{{{{{{\rm{Cl}}}}}}}}^{-}}\)=1.25). A total of n=6 recordings similar to (a) from 2 different precursor δ-LIT preparations have been conducted. b With symmetric KCl and added asymmetric calcium the precursor δ-LIT channel displays a Vrev = +20 mV, characteristic now for a highly Ca2+ selective channel (\({P}_{{{{{{{{\rm{Ca}}}}}}}}^{2+}}/{P}_{{{{{{{{\rm{Cl}}}}}}}}^{-}}=18\)), demonstrating a dramatic property change induced by the presence of Ca2+ ions. A total of n = 6 recordings similar to (b) from two different precursor δ-LIT preparations have been conducted. c The current–voltage relation of the mature δ-LIT is again linear but with low current noise and frequent, defined gating from the open towards the closed state. (displayed in extension box). d Next trans chamber perfusion within the same experiment. The Vrev = 7 mV value discloses cation selective properties \(({P}_{{{{{{{\rm{K}}}}}}}^{+}}/{P}_{{{{{{{{\rm{Cl}}}}}}}}^{-}} = 1.47)\) preserved within the mature variant. A total of n = 6 recordings similar to (c, d) from two different δ-LIT preparations have been conducted. With asymmetric KCl and cis added calcium (lower panel, no calcium addition trans) the precursor δ-LIT channel displays an asymptotic sine current–voltage relation with a zero-current crossing at Vrev = 0 mV. Extrapolation of the tangent to zero net current yields \({V}_{{{{{{{\rm{rev}}}}}}}}=\approx 40{{{{{{\rm{mV}}}}}}}\) and a very high calcium selectivity of the mature δ-LIT channel (\({P}_{{{{{{{{\rm{Ca}}}}}}}}^{2+}}/{P}_{{{{{{{\rm{K}}}}}}}^{+}}{/P}_{{{{{{{{\rm{Cl}}}}}}}}^{-}}\cong 600/1.47/1\)). A total of n ≥ 6 recordings similar to (d; lower panel) from 2 different δ-LIT preparations have been conducted. e Calculated GHK current–voltage relation using the experimental relative permeabilities from (d; lower panel) and the experimental concentration of \({{{{{{{\rm{Ca}}}}}}}}^{2+}\),\({{{{{{\rm{K}}}}}}}^{+}\) and \({{{{{{{\rm{Cl}}}}}}}}^{-}\)ions in the cis and trans compartment (see Supplemental Fig. 12 for details).
