# Using Small Compounds as Probes for Studying Mechanosensitive Channel Gating

> **NIH NIH R01** · UT SOUTHWESTERN MEDICAL CENTER · 2020 · $315,865

## Abstract

Project Summary/Abstract
One of the main reasons to study the bacterial mechanosensitive channel of large conductance, MscL, is that it
has, and will continue to serve as a molecular paradigm for the investigation of mechanosensory transduction.
With a crystal structure of what appears to be a ‘nearly-closed’ state of M. tuberculosis MscL, the channel has
advanced the field considerably by allowing researchers to overlay genetic and molecular analyses, coupled
with electrophysiology, onto a structural model. Thus, MscL from E. coli (Ec-MscL), which was the first defini-
tive mechanosensitive channel identified, continues to serve as a tractable model for determining principles for
how a protein senses and responds to membrane tension. However, another reason to study MscL is that the
channel serves a vital function in maintaining osmotic homeostasis of microbes. It normally serves as a biolog-
ical emergency release valve; upon osmotic downshock it opens a huge 30Å pore that allows for the rapid re-
lease of many accumulated cytoplasmic components, including potassium and glutamate, thus preventing cell
lysis. When the channel gates inappropriately it can lead to the death of the microbial cell; it thus is a viable
pharmacological target for potential antibiotics. Historically, one of the limitations in the study of MscL function
and pharmacology has been the total lack of small molecular probes that bind and modulate the channel. From
a High Throughput Screening (HTS) facility on campus, we have identified 18 novel chemical compounds that
inhibit the growth of E. coli in a MscL-dependent manner; surprisingly, an additional hit was streptomycin,
which appears to directly bind to and increase the probability of opening the MscL channel. We have used this
system to develop and refine assays for determining compound efficacy. In 96 well plates we can assay cell
growth to determine the minimal inhibitory concentrations (MIC) of compounds in the presence or absence of
expressed Ec-MscL, MscL orthologues, or the unrelated bacterial mechanosensitive channel MscS as a nega-
tive control. In addition, we have potassium and glutamate flux assays to measure the results of MscL gating in
vivo, the ability to determine channel function by electrophysiology, and have developed assays to determine
the binding sites of compounds. We will use this array of assays to determine if and how the novel compounds
identified in the HTS bind and modulate MscL activity. These studies will yield insight into mechanosensitive
channel gating mechanisms; in addition, co-crystallization of MscL with one or more of these compounds may
yield an open state structure for MscL, and the findings could eventually lead to a new generation of antibiotics.

## Key facts

- **NIH application ID:** 10001541
- **Project number:** 5R01GM121780-04
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** PAUL BLOUNT
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $315,865
- **Award type:** 5
- **Project period:** 2017-09-19 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10001541

## Citation

> US National Institutes of Health, RePORTER application 10001541, Using Small Compounds as Probes for Studying Mechanosensitive Channel Gating (5R01GM121780-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10001541. Licensed CC0.

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