Abstract
Chloride intracellular channels (CLIC) are non-classical ion channels lacking a signal sequence for membrane targeting. In eukaryotes, they are implicated in cell volume regulation, acidification, and cell cycle. CLICs resemble the omega class of Glutathione S-transferases (GST), yet differ from them in their ability to form ion channels. They are ubiquitously found in eukaryotes but no prokaryotic homolog has been characterized. We found that indanyloxyacetic acid-94 (IAA-94), a blocker of CLICs, delays the growth of Escherichia coli. In silico analysis showed that the E. coli stringent starvation protein A (SspA) shares sequence and structural homology with CLICs. Similar to CLICs, SspA lacks a signal sequence but contains an omega GST fold. Electrophysiological analysis revealed that SspA auto-inserts into lipid bilayers and forms IAA-94-sensitive ion channels. Substituting the ubiquitously conserved residue leucine 29 to alanine in the pore-forming region increased its single-channel conductance. SspA is essential for cell survival during acid-induced stress, and we found that acidic pH increases the open probability of SspA. Further, IAA-94 delayed the growth of wild-type but not sspA null mutant E. coli. Our results for the first time show that CLIC-like proteins exist in bacteria in the form of SspA, forming functional ion channels.
Highlights
The chloride intracellular channel (CLIC) proteins are well conserved in most eukaryotic species[1]
We further explore biophysical properties of stringent starvation protein A (SspA) by structure-function studies and show that similar to Chloride intracellular channels (CLIC), SspA channel currents are blocked by a known CLIC protein blocker, indanyloxyacetic acid-94 (IAA-94), reinforcing the idea that SspA proteins are related to CLICs
The addition of IAA-94 in the culture medium delayed the growth of E. coli at 37 °C, which was directly dependent on the dose of IAA-94 (Fig. 1A)
Summary
Shubha Gururaja Rao[1], Devasena Ponnalagu[1], Sowmya Sukur[1], Harkewal Singh[2], Shridhar Sanghvi1,Yixiao Mei[1], Ding J. CLICs resemble the omega class of Glutathione S-transferases (GST), yet differ from them in their ability to form ion channels They are ubiquitously found in eukaryotes but no prokaryotic homolog has been characterized. The founding member of CLICs, p64 was purified from bovine kidney extracts through its affinity to a chloride current blocker, indanyloxy acetic acid 94 (IAA-94)[2] They are a unique class of ion channels with a property to exist as both soluble and membrane forms[1, 3,4,5]. We have characterized the bacterial CLIC homolog, SspA, determined its biophysical properties as an ion channel, shown that it forms an acid-sensitive channel and its expression is essential for IAA-94 mediated reduction in growth
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