Thursday, July 6, 2017

Universal gating mechanism for TRPM class of ion channels?

We have discussed the implications of our findings from a recent publication and a scientifically unethical publication confirming one of the discoveries reported therein at length in other posts. One additional finding from our paper which is yet to receive any attention is the discovery of a new member of the SLOG superfamily found fused to the well-studied yet still in many ways poorly-understood Transient Receptor Potential Melastatin family (TRPM) of ion channels. Our research points to a likely regulatory role for this newly-discovered SLOG domain in TRPM channels, possibly by functioning in a universal gating mechanism for this clade of channels.

TRPM channels are usually involved in the transmembrane passage of monovalent ions, but have also been shown to transport divalent cations with varying specificity. Eight distinct paralogs (TRPM1-8) have been recognized in mammals, previous studies have traced individual paralogs to distinct starting points during animal evolution, ultimately pointing to their common origin in choanoflagellates. Studies devoted to understanding TRPM1-8 have reported widely varying tissue expression distribution patterns, gating properties, and functional roles while being linked to a range of human disease conditions including, but not limited to, hypomagnesia with secondary hypocalcemia (TRPM6), Guamanian amyotrophic lateral sclerosis/Parkinsonism dementia (TRPM7), and cardiovascular disease (TRPM7).

As is frequently observed in other classes of ion channels, several distinct protein domains have been identified in TRPMs, adorning the central transmembrane region which forms the channel. Prior to our recently-published study, the known core architecture shared across TRPM proteins proceeded as follows: a large N-terminal region with no previously known homology to any other domain, followed by a 6 TM helix-containing region forming the ion channel and the so-called “TRP-box” motif. Additionally, the TRPM2 protein is C-terminally fused to a NUDIX domain while the TRPM6/7 proteins are C-terminally fused to a Protein Kinase domain (see figure 1).

Figure 1

Deletion studies of TRPM channels had previously determined that the large N-terminal region was essential for ion channel function, although the presence of any specific protein domain units in this region had eluded researchers. At least one study had specifically linked this N-terminal region to a role in channel activation, and many of the SNPs associated with the disease conditions above mapped to this same N-terminal region. It was within this region that we observed the presence of the novel SLOG domain, which we dubbed the “LSDAT” family of SLOG domains. This discovery prompted further investigation into this region, and we were able to determine the complete domain architectures of TRPM channels: the SLOG domain is followed by three divergent Ankyrin repeats (a fusion shared with other families of TRP channels) before leading into the previously-described core architecture (see Figure 1). Additionally, several TRPM channels were further fused to a C-terminal cysteine-rich region. Delineation of the core architecture (SLOG+3 Ankyrin+ion-channel) led to novel evolutionary insights into the TRPM channels: beyond the versions identified in animals and choanoflagellates, the core TRPM architecture is also present in algae such as the cryptomonad Guillardia and the haptophyte Emiliania. This suggests a potentially deeper evolutionary origin for the TRPM proteins than previously thought. Versions of the LSDAT SLOG domains were also identified in ciliate genomes. While these often exist as standalone domains, on occasion they are found fused to a distinct ion channel or Ras-like GTPase domains, suggesting multiple independent recruitment events of the LSDAT domain to distinct transmembrane channels in eukaryotes.

The samepaper published by our group provides the first overview of the SLOG domain superfamily, which is a Rossmannoid domain most closely related to the deoxyribohydrolase (DRHyd) and TIR superfamilies to the exclusion of all other Rossmannoid domains and feature a shared atypical nucleotide binding pocket. We observed that SLOG domains appear to function in one of four general roles in the cell: 1) as an enzyme which modifies nucleotides via a base-clipping reaction (as is seen in the classical LOG family which generates cytokinin and cytokinin-like molecules in plants and some bacteria); 2) as a single-stranded DNA-binding protein (Smf/DprA); 3) as a sensor or processor of nucleotides in conflict systems which are centered on the production of a nucleotide intermediate; 4) or as a probable regulator of transmembrane domains, predicted as such due to their genomic association with various distinct transmembrane domains.

The LSDAT SLOG domain falls into the latter category: it is most closely related to a family of bacterial SLOG domains whose lone conserved genomic association is the fusion or adjacent positioning in the genome to the SLATT transmembrane domain family which are predicted to function as potential membrane pore-forming effectors. In some bacteria this system might have been “domesticated” to constitute a system where the SLOG and SLATT domains might work cooperatively as a signaling system with the SLOG domain regulating the formation of or flux through the transmembrane pore constituted by the transmembrane helices of the SLATT domain. This could involve a gating mechanism controlled by the binding and/or processing of a nucleotide ligand by the SLOG domain with additional interactions via the C-terminal cytoplasmic region of the SLATT domain (Figure 2).

In the case of the LSDAT, residue conservation patterns observed through multiple sequence alignment construction suggest there is unlikely to be any enzymatic activity; however, the key residues involved in structuring the atypical Rossmannoid binding pocket shared between the SLOG-DRHyd-TIR superfamilies and the residues likely to be involved in contacting a nucleotide ligand are retained. The sum of these observations led us to predict a role for LSDAT in ligand-binding which influences TRPM membrane channel activity. A universal TRPM ligand has never been identified; instead, a range of distinct small molecule ligands have been linked to gating and regulation of the channels including ADP-ribose, cyclic ADP-ribose (cADPR), NAADP, cAMP, H2O2, and phosphatidylinositol (4,5)-bisphosphate (PIP2). ADP-ribose, cADPR and NAADP are generally believed to act via the C-terminal cytoplasmic Nudix domain found in TRPM2 channels. In light of our discovery of the LSDAT SLOG domain in all TRPM channels, it appears very likely that nucleotide-derived ligands are more generally involved in TRPM channel regulation. It remains possible that these domains recognize such a universal ligand which gates ion transport in TRPM channels.