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The Gloeobacter ligand-gated ion channel (GLIC) is a bacterial homolog of vertebrate Cys-loop ligand-gated ion channels. Its pore-lining region in particular has a high sequence homology to these related proteins. Here we use electrophysiology to examine a range of compounds that block the channels of Cys-loop receptors to probe their pharmacological similarity with GLIC. The data reveal that a number of these compounds also block GLIC, although the pharmacological profile is distinct from these other proteins. The most potent compound was lindane, a GABAA receptor antagonist, with an IC50 of 0.2 μM. Docking studies indicated two potential binding sites for this ligand in the pore, at the 9′ or between the 0′ and 2′ residues. Similar experiments with picrotoxinin (IC50 = 2.6 μM) and rimantadine (IC50 = 2.6 μM) reveal interactions with 2′Thr residues in the GLIC pore. These locations are strongly supported by mutagenesis data for picrotoxinin and lindane, which are less potent in a T2′S version of GLIC. Overall, our data show that the inhibitory profile of the GLIC pore has considerable overlap with those of Cys-loop receptors, but the GLIC pore has a unique pharmacology.

The genome of thylakoidless cyanobacterium Gloeobacter violaceus encodes a fast-cycling rhodopsin capable of light-driven proton transport. We characterize the dark state, the photocycle, and the proton translocation pathway of GR spectroscopically. The dark state of GR contains predominantly all-trans-retinal and, similar to proteorhodopsin, does not show the light/dark adaptation. We found an unusually strong coupling between the conformation of the retinal and the site of Glu132, the homolog of Asp96 of BR. Although the photocycle of GR is similar to that of proteorhodopsin in general, it differs in accumulating two intermediates typical for BR, the L-like and the N-like states. The latter state has a deprotonated cytoplasmic proton donor and is spectrally distinct from the strongly red-shifted N intermediate known for proteorhodopsin. The proton uptake precedes the release and occurs during the transition to the O intermediate. The proton translocation pathway of GR is similar to those of other proton-pumping rhodopsins, involving homologs of BR Schiff base proton acceptor and donor Asp85 and Asp96 (Asp121 and Glu132). We assigned a pair of FTIR bands (positive at 1749 cm−1 and negative at 1734 cm−1) to the protonation and deprotonation, respectively, of these carboxylic acids.

Gloeobacter violaceus PCC 7421 is a slow-growing cyanobacterium which lacks thylakoid membranes, but whose five-membered psbA gene family encodes three isoform variants of the PsbA (D1) reaction center protein of Photosystem II. Under standard culture conditions Gloeobacter exhibits photosystem II electron transport, but several clear modifications in the redox potential of key cofactors bound by the PsbA protein are manifested in the flash-fluorescence characteristics. In other cyanobacteria dynamic expression of multiple psbA genes and turnover of PsbA isoforms is critical to counter excitation stress. We found that each of Gloeobacter's five psbA genes is expressed, with transcript abundances spanning 4.5 orders of magnitude. psbAI (glr2322) and psbAII (glr0779), encoding identical PsbA:2 form proteins, are constitutively expressed and dominate the psbA transcript pool under control conditions. psbAIII (gll3144) was strongly induced under photoinhibitory high irradiance stress, thereby contributing to a large increase in the psbA transcript pool that allowed cells to maintain their PsbA protein pools and then recover from irradiance stress, within one cellular generation. In contrast, under comparable photoinhibition provoked by UVB the cells were unable to maintain their psbA transcript and PsbA protein pools, and showed limited subsequent recovery. psbAIV (glr1706) and psbAV (glr2656), encoding two divergent PsbA isoforms, showed consistent trace expression but were never quantitatively significant contributors to the psbA transcript pool.