High Affinity Block of ICl,swell by Thiol-Reactive Small Molecules

Author

Sung H. Park, Virginia Commonwealth UniversityFollow

DOI

https://doi.org/10.25772/ACX8-TR69

Defense Date

2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Physiology and Biophysics

First Advisor

Dr. Clive M. Baumgarten

Abstract

Ebselen (Ebs) is considered as a glutathione peroxidase (GPx) mimetic and primarily thought to function by scavenging intracellular reactive oxygen species (ROS). Previous to our work, Deng et al. (2010a) demonstrated complete block of I_Cl,swell with 15 microM Ebs following endothelin-1 (ET-1) induced activation of the current in cardiomyocytes. This block was presumed to take effect mainly via the quenching of ROS. Nonetheless, our work with DI TNC1 astrocytes strongly emphasizes that Ebs might function by an alternative mechanism based on its kinetic profile in blocking I_Cl,swell. Our experiments showed that 45 nM Ebs can fully block I_Cl,swell thus suggesting an apparent IC₅₀ result, we predicted Ebs to possess a high k_on with a low k_off close to zero. As predicted, Ebs failed to washout in the timescale covered by our patch-clamp experiments. The block was also distal to H₂O₂, previously considered as the most proximate regulator of I_Cl,swell. And based on further evidence demonstrating irreversible block of I_Cl,swell distal to H₂O₂ with Ebs congeners, complete suppression of native I_Cl,swell with MTS reagents, and failure of Ebs to block I_Cl,swell from the cytosol, we concluded that Ebs and its congeners can covalently modify important –SH groups required for current activation while functioning as sulfhydryl reagents. Complete irreversible block of I_Cl,swell with 110 mM cell impermeant MTSES in native DI TNC1 astrocytes contrasts sharply to SWELL1 (Qiu et al., 2014) or LRRC8A (Voss et al., 2014), the latest molecular entity presumably responsible for I_Cl,swell, where 3.33 mM MTSES failed to demonstrate block of I_Cl,swell in the wild-type stably expressing SWELL1 (Qiu et al., 2014). Our data with Ebs, its congeners, and MTS reagents indicate the existence of a common extracellular binding site which involves a selenenylsulfide (Se-S) bond that critically modulates I_Cl,swell. We, therefore, synthesized a derivative of Ebs called ebselen-para-yne (Ebs-p-yne), which provided an even higher affinity for blocking I_Cl,swell with a presumed IC₅₀ ~picomolar range. Ebs-p-yne is a promising novel molecule that may serve as a tag in identifying the molecular fingerprint ultimately responsible for I_Cl,swell. Furthermore, we can take advantage of click chemistry to ultimately pull out the channel or channel component which has remained elusive for greater than two decades.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-8-2016