Triplin: Functional Probing of Its Structure and the Dynamics of the Voltage-Gating Process

Abstract

Gram-negative bacteria have a large variety of channel-forming proteins in their outer membrane, generally referred to as porins. Some display weak voltage dependence. A similar trimeric channel former, named Triplin, displays very steep voltage dependence, rivaling that responsible for the electrical excitability of mammals, and high inter-subunit cooperativity. We report detailed insights into the molecular basis for these very unusual properties explored at the single-molecule level. By using chemical modification to reduce the charge on the voltage sensors, they were shown to be positively charged structures. Trypsin cleavage of the sensor eliminates voltage gating by cleaving the sensor. From asymmetrical addition of these reagents, the positively charged voltage sensors translocate across the membrane and are, thus, responsible energetically for the steep voltage dependence. A mechanism underlying the cooperativity was also identified. Theoretical calculations indicate that the charge on the voltage sensor can explain the rectification of the current flowing through the open pores if it is located near the pore mouth in the open state. All results support the hypothesis that one of the three subunits is oriented in a direction opposite to that of the other two. These properties make Triplin perhaps the most complex pore-forming molecular machine described to date.

Triplin: Mechanistic Basis for Voltage Gating

“…Pore closure is proposed to result from the entry of the sensor into the pore, thus blocking ion flow. When the sensor is in the pore, it is proposed to interact with a negative domain located at the other end of the pore, with the negative domain also being responsible for the pore's weak cation selectivity and rectification [1]. No evidence was presented to support this closing mechanism, and this paper addresses that issue.…”

“…Evidence indicates that the sensors responsible for the closure of the Triplin pores must contain a minimum of 14 positive charges and most of those are likely to be arginines [1]. If, as proposed, a sensor were to physically block a pore by inserting into the pore, it is reasonable to conclude that evolutionary adaptation of the process would result in a binding surface that complements the physical nature of the sensor.…”

“…Triplin is a channel-forming structure found in Escherichia coli. 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.…”

“…Insights into the molecular basis for this complex were obtained by probing the nature and dynamics of the voltage sensors of Triplin [1]. This led the authors to propose the following model.…”

“…Triplin is proposed to form three beta barrel pores, similar to those of other three-pore porins, but one pore-forming structure is oriented opposite to the others [1]. In the model (Figure 2), each pore-forming subunit has a positively charged voltage sensor (blue region) located in external loops on one end of the pore.…”