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).
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| 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.
