Cooperativity and Steep Voltage Dependence in a Bacterial Channel

Abstract

This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel channel-former, isolated from Escherichia coli, is consistent with a linearly organized three-channel unit displaying steep voltage-gating (a minimum of 14 charges in the voltage sensor) that rivals that of channels in mammalian excitable membranes. The channels also display strong cooperativity in that closure of the first channel permits the second to close and closure of the second channel permits closure of the third. All three have virtually the same conductance and selectivity, and yet the first and third close at positive potentials whereas the second closes at negative potentials. Thus, is it likely that the second channel-former is oriented in the membrane in a direction opposite to that of the other two. This novel structure is named “triplin.” The extraordinary behavior of triplin indicates that it must have important and as yet undefined physiological roles.

Triplin: Mechanistic Basis for Voltage Gating

“…The kinetics of a well-studied beta barrel channel former, VDAC, provide useful contrasting kinetic properties to Triplin. For Triplin, the rates of pore closure for pores 2 and 3 are fast whereas the rates of reopening are slow [6]. For VDAC, pore closure is slow (τ ≈ 400 s) and voltage dependent whereas reopening is fast (τ ≈ 2.5 ms) and voltage independent [8].…”

“…Triplin is a three-pore complex, each pore possessing a positively charged voltage sensor consisting of 14 net charges [1,6]. Each sensor domain translocates across the membrane in a steeply voltage-dependent manner, and this translocation is coupled with the closure of each pore [1].…”

“…The similarity of the conductance and selectivity of the Triplin pores to those of porins like OmpF and OmpC whose structure is well established, makes the assumption of a beta barrel very likely to be correct. The steep voltage dependence of the Triplin pores, requiring the translocation of 14 charges across the membrane [6], only leaves 2 mechanisms: translocation of charges through the lipid bilayer or translocation through the hole formed by the pore. Other models for the gating of porins, such as the movement of a loop located withing the pore itself, are not tenable.…”

“…This membrane is identical to a natural cell membrane but lacks proteins or carbohydrates. Samples containing Triplin were generated as previously described [6], i.e., flash-frozen in 0.1 mL aliquots and stored at −80 • C. After thawing, β-octyl-glucoside was added to a final concentration of 1% (w/v) and kept on ice during the experiment. Typically, 10 µL of the sample was dispersed into one aqueous compartment (designated "cis"), and, with time, Triplin would insert into the membrane.…”

“…It forms a set of three pores whose effective size and conductance resemble OmpC, whereas its weak ion selectivity resembles OmpF [1]. Triplin differs dramatically for all porins described to date (e.g., [2][3][4][5]) in that it displays steep voltage dependence [6] comparable to that of the voltage-gated channels responsible for the electrical excitability of the mammalian nervous system. A unique property of Triplin that distinguishes it from all membrane channels and pores described to date is the remarkable interpore cooperative behavior in that pore 1 must close first, its closure then allows pore 2 to close and, in turn, pore 2 closure allows pore 3 to close [6].…”