A Bacterial Homolog of Chloride Intracellular Channel (CLIC) Protein Family, Stringent Starvation Protein A (SspA), forms a Non-Selective Ion Channel

Chloride Intracellular Channel (CLIC) proteins are non-canonical ion channels without a signal sequence targeting them to a membrane or transmembrane domain. CLICs exist insoluble and membranous forms, and are structurally similar to the omega class of Glutathione S transferases (GST). We have earlier shown that CLICs can auto-insert into planar bilayers to form trans-redox regulated ion channels. To identify a prokaryotic CLIC homolog, insilico and electrophysiological analysis were performed. Here we report a class of proteins called Stringent Starvation Protein A (SspA) in prokaryotes, which have a sequence and structural homology to CLIC proteins. Analysis of crystal structures of soluble forms of SspA and CLICs show omega folds and homologues α-helices and β-sheets. We further tested whether SspA can autoinsert to form functional ion channels. Recombinant SspA from E.coli was reconstituted in a planar bilayer made of POPE: POPS: Cholesterol with 4:1:1 molar ratios. SspA insert into planar bilayers and form functional ion channels within 5 minutes of addition of protein to the cis-side. These ion channels demonstrated single-channel conductance of 9.0±1.5 pS (n=5) and were more selective for cations (K+) than anions (Cl-), a property similar to CLIC. When SspA was recorded in Tris-Cl, single-channel conductance was lowered to 5.5±0.5 pS, with increased selectivity for anions over cations. IAA-94, a known CLIC blocker, decreased open probability of SspA-mediated currents (n=3). We have identified and characterized the first bacterial homolog of mammalian CLICs and shown it shares similar biophysical properties with CLIC proteins.