Markov chain Monte Carlo based analysis of post-translationally modified VDAC gating kinetics.

Shivendra G Tewari,Daniel A Beard,Ranjan K Dash,Bradley J Otto,Yifan Zhou,Wai-Meng Kwok

doi:10.3389/fphys.2014.00513

Abstract

The voltage-dependent anion channel (VDAC) is the main conduit for permeation of solutes (including nucleotides and metabolites) of up to 5 kDa across the mitochondrial outer membrane (MOM). Recent studies suggest that VDAC activity is regulated via post-translational modifications (PTMs). Yet the nature and effect of these modifications is not understood. Herein, single channel currents of wild-type, nitrosated, and phosphorylated VDAC are analyzed using a generalized continuous-time Markov chain Monte Carlo (MCMC) method. This developed method describes three distinct conducting states (open, half-open, and closed) of VDAC activity. Lipid bilayer experiments are also performed to record single VDAC activity under un-phosphorylated and phosphorylated conditions, and are analyzed using the developed stochastic search method. Experimental data show significant alteration in VDAC gating kinetics and conductance as a result of PTMs. The effect of PTMs on VDAC kinetics is captured in the parameters associated with the identified Markov model. Stationary distributions of the Markov model suggest that nitrosation of VDAC not only decreased its conductance but also significantly locked VDAC in a closed state. On the other hand, stationary distributions of the model associated with un-phosphorylated and phosphorylated VDAC suggest a reversal in channel conformation from relatively closed state to an open state. Model analyses of the nitrosated data suggest that faster reaction of nitric oxide with Cys-127 thiol group might be responsible for the biphasic effect of nitric oxide on basal VDAC conductance.

Highlights

The mitochondrial inner membrane (MIM) and mitochondrial outer membrane (MOM) are the two phospholipid membranes separating the mitochondrial matrix milieu from cytosol
Because voltage-dependent anion channel (VDAC) has been linked to both cell survival and apoptosis, it has been described as the “gatekeeper of mitochondrial function and dysfunction (Kerner et al, 2012).”
We applied the generalized continuous-time Markov chain Monte Carlo (MCMC) method, using the Markov model shown in Figure 1A, for the electrophysiological recordings of WT, nitrosated, un-phosphorylated and phosphorylated VDAC activity

Summary

Introduction

The mitochondrial inner membrane (MIM) and mitochondrial outer membrane (MOM) are the two phospholipid membranes separating the mitochondrial matrix milieu from cytosol. The most abundant protein in the mammalian MOM is a voltage-dependent anion channel (VDAC) (Krimmer et al, 2001; Bayrhuber et al, 2008; Rostovtseva and Bezrukov, 2008). VDAC is the main conduit that connects the cytosol to the mitochondrial inter-membrane space (MIS; the narrow region between MOM and MIM), and allows the passage of carbon substrates (e.g., pyruvate, succinate), phosphates (e.g., ATP, ADP, Pi), and cations (e.g., Ca2+, K+, H+) across the MOM. Because VDAC has been linked to both cell survival and apoptosis, it has been described as the “gatekeeper of mitochondrial function and dysfunction (Kerner et al, 2012).”

Methods

Results

Conclusion