Fig. 1.

Tubulin at nanomolar concentrations induces reversible partial blockage of VDAC. (A) Representative current record through a single channel before and after addition of 50 nM tubulin to the cis side at −25 mV applied voltage. Addition of tubulin induces time-resolved rapid events of partial channel blockages to a single well-defined conductance level, shown in the Inset at a finer scale. Here and elsewhere, dashed lines indicate zero-current level; current records were filtered by using averaging times of 10 ms (except for 1 ms in Inset). (B) Typical VDAC voltage gating in tubulin-free solution under periodically applied −50 mV voltage impulses. Under applied voltage, channel conductance moves from a single high-conducting open state to various low-conducting closed states. Relaxing the voltage to 0 mV reopens the channel. The dashed-and-dotted line indicates the conductance of the channel in its open state and dotted lines in its closed states. (C) Tubulin induces a single well-resolved closed state of VDAC, whereas in control voltage-induced gating a wide variety of closed states is observed. The distribution of conductances in control was collected from a series of −50 mV impulses of 30 s duration interrupted by 70 s periods of zero applied voltage to restore the open state. (D) Statistical analysis of the tubulin-induced closures of VDAC performed by logarithmic exponential fitting of the open times (τ_on) and closed times (τ_off). It is seen that open time is described by single exponent with characteristic time τ_on = 198.7 ± 3.5 ms. Closed-time histogram could be fitted by at least 2 exponents with characteristic times of τ_off⁽¹⁾ = 2.4 ± 0.1 ms and τ_off⁽²⁾ = 30.4 ± 5.7 ms. The time histograms were collected from the current traces presented in A. Bilayer membranes were formed from the mixture of asolectin and cholesterol (10:1 wt/wt). VDAC was isolated from rat liver mitochondria. The medium consisted of 1 M KCl buffered with 5 mM Hepes at pH 7.4.