Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states
Abstract
In-silico and in-vitro studies have made progress in understanding protein-protein complexes formation; however, the molecular mechanisms for their dissociation are unclear. Protein-protein complexes, lasting from microseconds to years, often involve induced-fit, challenging computational or kinetic analysis. Charybdotoxin (CTX), a peptide from the Leiurus scorpion venom, blocks voltage-gated K+-channels in a unique example of binding/unbinding simplicity. CTX plugs the external mouth of K+-channels pore, stopping K+-ion conduction, without inducing conformational changes. Conflicting with a tight binding, we show that external permeant ions enhance CTX-dissociation, implying a path connecting the pore, in the toxin-bound channel, with the external solution. This sensitivity is explained if CTX wobbles between several bound conformations, producing transient events that restore the electrical and ionic trans-pore gradients. Wobbling may originate from a network of contacts in the interaction interface that are in dynamic stochastic equilibria. These partially-bound intermediates could lead to distinct, and potentially manipulable, dissociation pathways.
Data availability
Data used for Figures 2 to 7 is available in dryad.org
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Data from: Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional statesDryad Digital Repository, doi:10.5061/dryad.0p77qk4.
Article and author information
Author details
Funding
Fondo Nacional de Desarrollo Científico y Tecnológico (3160321)
- Hans Moldenhauer
Fondo Nacional de Desarrollo Científico y Tecnológico (3170599)
- Ignacio Díaz-Franulic
Fondo Nacional de Desarrollo Científico y Tecnológico (1171155)
- Horacio Poblete
Ministerio de Economía, Fomento y Turismo (MiNICAD)
- Horacio Poblete
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Leon D Islas, Universidad Nacional Autónoma de México, Mexico
Version history
- Received: February 18, 2019
- Accepted: July 4, 2019
- Accepted Manuscript published: July 4, 2019 (version 1)
- Version of Record published: July 26, 2019 (version 2)
Copyright
© 2019, Moldenhauer et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above—which were used to identify endogenous PANX1 phosphorylation at these two sites—are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.
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