Výsledky projektu Lokalizace a charakterizace vazebných míst pro Ca2+ vázající proteiny a fosfatidyl inositol fosfáty na intracelulárních koncích receptoru TRPM4

ABSTRACT:
Transient receptor potential melastatin-1 (TRPM1) is a calciumchannel that is essential for the depolarization of
photo-responsive retinal bipolar cells, but most of the physiological functions and cellular roles of this channel
are still poorly understood. Most transient receptor potential (TRP) channels are typically regulated by intracellular
proteins and other signalingmolecules. Phosphatidylinositol-4,5 bisphosphate (PIP2), a minor phospholipid
component of cellmembranes, has previously been shown to directly bind TRP channels and to play a unique role
inmodulating receptor function. To characterize the binding of PIP2 as a potential regulator of TRPM1, weutilized
biophysical methods and molecular modeling to study the interactions of PIP2 with an N-terminal fragment of
TRPM1 (residues A451–N566). The basic N-terminal residue K464 of TRPM1 suggests that it is part of putative
pleckstrin homology (PH) domain and is involved in the interactions with PIP2. This is the first report detailing
the binding of PIP2 at the N-terminus of the TRPM1 receptor.

ABSTRACT:
The transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective ion channel broadly
expressed in a variety of tissues. Receptor has been identified as a crucial modulator of numerous calcium
dependent mechanisms in the cell such as immune response, cardiac conduction, neurotransmission and insulin
secretion. It is known that phosphoinositide lipids (PIPs) play a unique role in the regulation of TRP channel
function. However the molecular mechanism of this process is still unknown. We characterized the binding
site of PIP2 and its structural analogue PIP3 in the E733–W772 proximal region of the TRPM4 N-terminus via
biophysical and molecular modeling methods. The specific positions R755 and R767 in this domain were
identified as being important for interactions with PIP2/PIP3 ligands. Their mutations caused a partial loss of
PIP2/PIP3 binding specificity. The interaction of PIP3 with TRPM4 channels has never been described before.
These findings provide new insight into the ligand binding domains of the TRPM4 channel.

Abstract
Transient receptor potential vanilloid 1 ion channel (TRPV1) belongs to the TRP family of ion channels. These channels play a role in many important biological processes such as thermosensation and pain transduction. The TRPV1 channel was reported to be also involved in nociception. Ca2+ ions are described to participate in the regulation of TRP channels through the interaction with Ca2+-binding proteins, such as calmodulin or S100A1. Calmodulin is involved in the Ca2+-dependent regulation of TRPV1 via its binding to the TRPV1 C-terminal region. However, the role of the Ca2+-binding protein S100A1 in the process of TRP channel regulation remains elusive. Here we characterized a region on the TRPV1 C-terminus responsible for the interaction with S100A1 using biochemical and biophysical tools. We found that this region overlaps with previously identified calmodulin and PIP2 binding sites and that S100A1 competes with calmodulin and PIP2 for this binding site. We identified several positively charged residues within this region, which have crucial impact on S100A1 binding, and we show that the reported S100A1-TRPV1 interaction is calcium-dependent. Taken together, our data suggest a mechanism for the mutual regulation of PIP2 and the Ca2+-binding proteins S100A1 and calmodulin to TRPV1.

Transient receptor potential kanál TRPC6 je neselektivní iontový kanál zodpovědný za vstup vápenatých kationtů do buňky. V této studii bylo pomocí fluorescenční anizotropie charakterizováno vazebné místo pro S100A1 protein na C-konci TRPC6.