I. Sequence diversity of tubulin isotypes in regulation of mitochondrial respiration The microtubule protein tubulin is a heterodimer comprising alpha/beta subunits, and each subunit features multiple isotypes in vertebrates. For example, the human proteome contains as many as seven alpha-tubulin and eight beta-tubulin isotypes. These isotypes vary mostly in the length and primary sequence of the disordered anionic C-terminal tails (CTTs). The biological reason for such sequence diversity remains a topic of vigorous enquiry. We now demonstrate that this diversity may be a key feature of tubulins role in regulating the permeability of the mitochondrial outer membrane voltage-dependent anion channel (VDAC). Previously, we have shown that in addition to their important functions involving interactions with motor proteins and other microtubule-associated proteins, tubulin CTTs are essential for tubulin interaction with VDAC. As the major transport channel and most abundant protein in the mitochondrial outer membrane, this channel is responsible for most of the metabolite flux in and out of mitochondria. VDAC was proven to be involved in a wide variety of mitochondria-associated pathologies, ranging from various forms of cancer to neurodegeneration. This year, using recombinant yeast alpha/beta-tubulin constructs with alpha-CTTs, beta-CTTs, or both from various human tubulin isotypes, we probed their interactions with VDAC reconstituted into planar lipid bilayers. A comparative study of the blockage kinetics revealed that both alpha-CTTs or beta-CTTs block VDAC pore, but the efficiency of blockage spans two orders of magnitude, depending on the CTT isotype. Beta-tubulin constructs, notably beta3, blocked VDAC most effectively. We quantitatively describe these experimental results using a physical model that accounts only for the number and distribution of charges in the CTT, and not for the interactions between specific residues on the CTT and VDAC pore. Based on these results, we speculate that the effectiveness of VDAC regulation by tubulin depends on the predominant tubulin isotype in a cell. Consequently, the fluxes of ATP/ADP through the channel could vary significantly depending on the isotype. Our results suggest an intriguing link between VDAC regulation and the diversity of tubulin isotypes present in vertebrates. II. Stochastic gating as a novel mechanism for channel selectivity In contrast to the highly ion-selective channels studied in neurophysiology, which have narrow selectivity filters of the size of a partially dehydrated ion, metabolite channels are significantly wider. Indeed, they have to accommodate metabolite molecules that typically are much larger than simple mono- or divalent ions. It is clear that the large pore size of metabolite channels jeopardizes one of the main membrane functions, namely, to serve as a barrier for solutes other than the particular metabolites these channels have evolved to pass. For example, VDAC, recognized to be the major pathway for ATP and ADP exchange between mitochondria and cytosol, is also highly permeable for molecules smaller than these metabolites. VDAC is also the most abundant integral protein of the outer mitochondrial membrane. However, interestingly, a number of studies have demonstrated that under physiological conditions these channels are predominantly closed, which allows the membrane to sustain its barrier function. This year, by applying our recent theory of the stochastic gating effect on channel-facilitated transport, we proposed that stochastic gating may provide a mechanism for metabolite channel selectivity in favor of slowly moving large solutes. Specifically, we showed that fast gating of a predominantly closed channel leads to an increase of the channel selectivity for large, slowly diffusing molecules versus small ions by orders of magnitude. We hypothesized that this, to our knowledge, newly described mechanism of the selectivity due to stochastic gating provides an explanation for the puzzling observation that VDAC in intact mitochondria is mostly closed. Our conjecture is that the large number of predominantly closed VDAC channels is necessary to keep ATP/ADP transport at a sufficiently high level while effectively suppressing small-ion leakage through the mitochondrial outer membrane. It is also worth mentioning that the stochastic gating effect considered by us might not be restricted to the VDAC case. We hypothesized that it can be used by Nature to minimize the shunting effect of other wide channels with respect to small solutes thus shedding light on functioning of different predominantly plugged beta-barrel channels (e.g., OprF of Pseudomonas aeruginosa) and on other biological processes controlled by passage through fluctuating bottlenecks. III. Lipid nanodomains in ion channel functioning The function of lipid rafts in mammalian cell membranes remains an issue of controversy, though the association of many membrane proteins with the relatively detergent-resistant membrane fraction of plasma membranes has been well established. However, to the best of our knowledge, the ability of nano-sized membrane inhomogeneities to modify channel behavior has not been yet established. This year, using raft-forming model membrane systems containing cholesterol, we show that lipid lateral phase separation at the nanoscale level directly affects the dissociation kinetics of the gramicidin dimer, a model ion channel. Gramicidin inserts into membranes and dimerizes to form cation selective ion channels. The conducting lifetime of these channels is exquisitely sensitive not only to lipid composition, but also to compounds that alter membrane mechanics. For a lipid raft model, we studied mixtures of dioleoylphosphatidylcholine, porcine brain sphingomyelin, and cholesterol. We found that the gramicidin channel detects the presence of separate nanoscale domains in the 1/1/1 mixture well above the microscopic miscibility transition as reported by fluorescence microscopy. Of particular interest for neurophysiology, we note that the proportion of cholesterol in nerve cell membranes is quite high, about 40% and that cholesterol depletion in live mammalian cells inhibits both raft nanodomain formation and activation of the (PI(3)K/Akt) pathway. Our findings finally demonstrate that nanodomains in cholesterol rich membranes do affect ion channel function. While the structure of gramicidin indeed differs from that of mammalian ion channels, many of the fundamental principles of ion conduction through channels were first demonstrated with gramicidin, and its sensitivity to the surrounding lipid environment is a property shared by a significant number of ion channels involved in synaptic signaling. Thus, we suggest that perturbations of lipid phase mixing produced by application of membrane active agents could affect nervous system function through the interactions at the nanoscale uncovered in our study.

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U.S. National Inst/Child Hlth/Human Dev
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Rostovtseva, Tatiana K; Gurnev, Philip A; Hoogerheide, David P et al. (2018) Sequence diversity of tubulin isotypes in regulation of the mitochondrial voltage-dependent anion channel. J Biol Chem 293:10949-10962
Berezhkovskii, Alexander M; Bezrukov, Sergey M (2018) Stochastic Gating as a Novel Mechanism for Channel Selectivity. Biophys J 114:1026-1029
Hoogerheide, David P; Gurnev, Philip A; Rostovtseva, Tatiana K et al. (2018) Real-Time Nanopore-Based Recognition of Protein Translocation Success. Biophys J 114:772-776
Berezhkovskii, Alexander M; Bezrukov, Sergey M (2018) Effect of stochastic gating on the flux through a membrane channel: a steady-state approach. J Phys Condens Matter 30:254006
Hoogerheide, David P; Gurnev, Philip A; Rostovtseva, Tatiana K et al. (2017) Mechanism of ?-synuclein translocation through a VDAC nanopore revealed by energy landscape modeling of escape time distributions. Nanoscale 9:183-192
Berezhkovskii, Alexander M; Dagdug, Leonardo; Bezrukov, Sergey M (2017) Mean Direct-Transit and Looping Times as Functions of the Potential Shape. J Phys Chem B 121:5455-5460
Hoogerheide, David P; Noskov, Sergei Y; Jacobs, Daniel et al. (2017) Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes. Proc Natl Acad Sci U S A 114:E3622-E3631
Gurnev, Philip A; Stanley, Christopher B; Aksoyoglu, M Alphan et al. (2017) Poly(ethylene glycol)s in Semidilute Regime: Radius of Gyration in the Bulk and Partitioning into a Nanopore. Macromolecules 50:2477-2483
Gurnev, Philip A; Roark, Torri C; Petrache, Horia I et al. (2017) Cation-Selective Channel Regulated by Anions According to Their Hofmeister Ranking. Angew Chem Int Ed Engl 56:3506-3509
Berezhkovskii, Alexander M; Dagdug, Leonardo; Bezrukov, Sergey M (2017) Bulk-mediated surface transport in the presence of bias. J Chem Phys 147:014103

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