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Published on: March 7, 2018
A Cell-Based Assay to Assess Hemichannel Function
Srinivasan Krishnan1, Mariana C Fiori1, Luis G Cuello1
1Department of Cell Physiology and Molecular Biophysics, and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX.
Insights
Researchers developed a novel assay to screen for drugs targeting connexin hemichannels, crucial in diseases like stroke and deafness. This simple, scalable method uses genetically modified bacteria to identify potential hemichannel inhibitors.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Connexin hemichannels are implicated in various pathologies, including deafness, stroke, and cardiac infarct, making them a significant therapeutic target.
- Current limitations in developing hemichannel-targeting drugs stem from a lack of selective inhibitors and efficient screening assays.
- The translational application of potential therapies is hindered by the non-selectivity and isoform non-specificity of existing inhibitors.
Purpose of the Study:
- To develop a novel, high-throughput screening assay for identifying drugs that modulate connexin hemichannel activity.
- To overcome the limitations of existing assays and facilitate the discovery of selective hemichannel inhibitors.
Main Methods:
- Genetically engineered a bacterial strain deficient in potassium (K+) uptake.
- Introduced functional human connexins into the modified bacterial strain.
- Utilized cell growth in low-K+ medium as a readout for functional hemichannel activity and K+ uptake.
Main Results:
- Demonstrated that the engineered bacteria exhibit growth in low-K+ medium only when functional connexin hemichannels are expressed.
- Established a simple, robust, and scalable cell-growth-based assay for assessing hemichannel function.
- Validated the assay's potential for high-throughput screening of potential hemichannel-active drugs.
Conclusions:
- The developed bacterial assay provides a powerful new tool for discovering and characterizing connexin hemichannel modulators.
- This assay facilitates the identification of selective inhibitors, paving the way for new therapeutic strategies for hemichannel-related disorders.
- The assay's scalability and simplicity offer a significant advancement in drug discovery for conditions linked to connexin hemichannel dysfunction.
Abstract:
Activation of connexin hemichannels is involved in the pathophysiology of disorders that include deafness, stroke, and cardiac infarct. This aspect makes hemichannels an attractive therapeutic target. Unfortunately, most available inhibitors are not selective or isoform specific, which hampers their translational application. The absence of a battery of useful inhibitors is due in part to the absence of simple screening assays for the discovery of hemichannel-active drugs. Here, we present an assay that we have recently developed to assess hemichannel function. The assay is based on the expression of functional human connexins in a genetically modified bacterial strain deficient in K+ uptake. These modified cells do not grow in low-K+ medium, but functional expression of connexin hemichannels allows K+ uptake and growth. This cell-growth-based assay is simple, robust, and easily scalable to high-throughput multi-well platforms.
