Ljaschenko, D. | Search bioRxiv

bioRxiv2023

Intron retention of an adhesion GPCR generates single transmembrane-helix isoforms to enable 7TM-adhesion GPCR function

Adhesion G protein-coupled receptors (aGPCR) function as metabotropic mechanosensors in the nervous system and other organs. aGPCR are heavily spliced forecasting an extraordinary molecular structural diversity. Many predicted isoforms lack the transmembrane (7TM) signaling subunit, but to what extent these non-GPCR isoforms are produced and what physiological purpose they serve is unknown. Alternative splicing through intron retention of ADGRL/Latrophilin/Cirl mRNA in Drosophila generates transcripts encoding unconventional proteins with an extracellular domain anchored by a single transmembrane helix (Cirl1TM). Here, we show that Cirl1TM transcripts are translated in vivo and that Cirl1TM binds Cirl7TM N-terminal fragment-dependently. This interaction enables mechanosensory neurons to distinguish input intensities through Go-dependent signaling. Similarly, a direct interaction was found for mammalian GPR126/ADGRG6 isoforms. Together, our findings define intron retention and isoform-specific heteromerization as extraordinary molecular strategies to adjust Cirl-dependent mechanosensation and demonstrate physiological relevance of versatile aGPCR isoform repertoire to tune cellular responsiveness.

neuroscience↗

bioRxiv2021

Channel opening duration in adult muscle nAChRs determined by activated external ACh binding site

We recorded currents through the cell membrane at single nAChR molecules, held at ACh or Epibatidine (Ebd) concentrations of 0.01, 0.1, 1, 10 or 100 {micro}M. The measured current amplitudes had an absolutely fixed value of 15 pA. This was valid for different agonists at all concentrations. Binding an agonist at one or both sites in the ring of subunits allowed to open the channel, the site that initiated the opening determined the duration of the final opening of the channel. In addition, the current flow was continuously interrupted by < 3 {micro}s shut times. The resolution of our records was optimized to reach 5 {micro}s, but was insufficient to resolve an unknown proportion of shorter shut times. Therefore, measured durations of openings are overestimated, and cited in brackets: {tau}o1 (3 {micro}s) elicited by agonist-binding at the {delta}-site, {tau}o2 and {tau}o3 (40 and 183 {micro}s) by binding at the {varepsilon}-site, and {tau}o4 (752 {micro}s) by binding at the {varepsilon}- and {delta}-site. Mono-liganded nAChRs trigger short bursts of 0.6 ms duration. Bi-liganded nAChRs generate long bursts that at low agonist concentrations last 12 ms. Above 10 {micro}M ACh, long bursts are shortened, with 100 {micro}M ACh, to 5 ms, and further at higher concentrations. While ACh was the main agonist, Ebd bound more effectively than ACh to the {varepsilon}-site. SIGNIFICANCETransmembrane currents were recorded from single nAChR molecules. Binding ACh at one of the external receptor sites opens the central current channel repeatedly for a period selected by the activated receptor site, and transmembrane current can flow with fixed 15 pA amplitude (same at various agonists and range of concentrations, unaffected by desensitization). Current flow is interrupted by fixed < 3 {micro}s blocks of the central channel that border each opening. Sets of current block and opening can be repeated for receptor-determined periods of time and form bursts of channel openings. The results may help to interpret the structural reactions in the center of the molecule that are addressed in the introduction.

neuroscience↗