| Autor: |
Nisha Shrestha, Samuel Kosydar, Daniel Fologea, Philip Belzeski, Lauren McDaid, Paul Carnig, Jason May, Sheenah Bryant |
| Rok vydání: |
2016 |
| Předmět: |
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| Zdroj: |
Biophysical Journal. 110(3) |
| ISSN: |
0006-3495 |
| DOI: |
10.1016/j.bpj.2015.11.3166 |
| Popis: |
Lysenin, a 297 amino acid pore forming toxin extracted from the earthworm E. foetida, inserts hexameric channels in natural and artificial membranes containing sphingomyelin. A distinct feature of lysenin channels is their asymmetric voltage induced gating which occurs at low positive transmembrane potentials. Although the structure of the channel is not yet solved, previous studies of the lysenin monomeric structure and interaction with membranes indicate the presence of local positive charges within the protein's structure. Consequently, we hypothesized that highly charged organic anions such as ATP, ADP, and AMP may interact with the channel and alter its functionality. Here we show that the macroscopic conductance of the channels was diminished by addition of various adenosine phosphates in a charge and concentration-dependent manner. Single channel explorations have shown a gradual reduction of the ionic currents indicating interaction with absence of gating. Buffer-exchange experiments demonstrated that the current inhibition is reversible, pointing out a potential electrostatic mechanism of interaction between channels and anions which implies binding. This hypothesis was further sustained by analyzing the inhibitory effects of ATP comprising ionic solutions with different ionic strengths. Hill analysis provided further insights into the inhibition process and indicated a positive cooperativity between binding events. In addition, the I-V plots recorded in the presence of increasing inhibitor concentration demonstrate a significant rightward shift of the voltage-induced gating profile. The reduced effects elicited by AMP as compared with ATP and ADP, the reversibility of the inhibition, and the strong dependency of the observed effects on the ionic strength point out an electrostatic mechanism of interaction which may yield partial occlusion of the channels and modulation of the voltage-induced gating. This work sets an example of purinergic signaling that implies modulating the functionality of an exogenous membrane transporter. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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