Figure 5.

α-Syn blocks hVDAC3 less efficiently than mVDAC1. (A) Representative hVDAC3 and mVDAC1 open single-channel current traces before and after the addition of 10 nM α-syn (at +30 mV of applied voltage). Insets show fragments of current records at a finer time scale. Current records were digitally filtered at 5 kHz using a low-pass Bessel (8-pole) filter. (B) Log-binned distributions of the open time τ_on obtained with 20 nM α-syn at +30 mV for hVDAC3 and mVDAC1 and acquired from statistical analysis of the corresponding current records. Solid lines are logarithmic single-exponential fittings with characteristic times <τ_on> equal to 107.3 and 6.1 ms for hVDAC3 and mVDAC1, respectively. Norm., normalized. (C) Voltage dependences of the on-rate constant k_on of α-syn binding to hVDAC3 and mVDAC1. The solid lines represent a fit to Boltzmann equation k_on(V) = k₀ exp [n_be|V|/(k_BT)], where V is the applied voltage, n_b is the effective binding charge of 2.6 ± 0.3, and e, k_B, and T have their usual meaning of elementary charge, Boltzmann constant, and absolute temperature, respectively. (D) Voltage dependences of the mean blockage time τ_off obtained for hVDAC3 and mVDAC1. The increase of τ_off with voltage amplitude corresponds to the α-syn blockage/retraction regime; the decrease of τ_off with voltage amplitude (highlighted in yellow) corresponds to the regime of α-syn translocation through the pore. V* indicates the voltage separating the two regimes. Data are means of three to four experiments ± SD. Membranes were formed from mixtures of DOPC/DOPE/2DOPG, and membrane bathing solutions contained 1 M KCl buffered with 5 mM HEPES at pH 7.4.