Scopul nostru este sprijinirea şi promovarea cercetării ştiinţifice şi facilitarea comunicării între cercetătorii români din întreaga lume.
Autori: Tudor Luchian
While G protein activated inwardly rectifying K”+ channels (GIRKs) are activated by binding of the #beta##gamma# subunits of an activated G protein, the molecular mechanism of gating of GIRKs by G_#beta#_#gamma# remains obscure. In order to elucidate the possible role of putative cytoplasmic regions of the GIRK1/GIRK5 channel in G protein- dependent and intrinsic gating of the channel, synthetic peptides derived from the GIRK1 sequence were designed. The peptides were tested in isolated inside- out patches from membranes of Xenopus laevis oocytes expressing GIRK1/GIRK5 (in oocytes injected with GIRK1 RNA) and GIRK1/GIRK4 channels, for their ability to modify gating behavior. A particular peptide (termed DS6) comprising the very end 17 aminoacids from the C- terminal tail of the GIRK1 subunit (TRMEGNLPAKLRKMNSD), turned to be a possible candidate for playing roles in the gating behavior of the channel. Single-channel experiments have proven that this peptide reversibly blocked GIRK activity with IC_5_0 values of 7.9 #+-# 2.0 or 3.5 #+-# 0.5 #mu#g ml”-“1 for GIRK1/GIRK5 or GIRK1/GIRK4 channels, respectively. Noteworthy, the DS6 peptide can work very effective in the case of heterooligomeric protein complexes which are closer related to those existing in-vivo, as well (GIRK1 subunits aggregate with GIRK4 ones in order to form a fully functional inwardly-rectifying K”+ channel). The interaction between the studied GIRK channels and the DS6 peptide does not entail any changes at all in the magnitude of the K”+ current mediated by the channels (clamped at -80 mV throughout), the only alteration induced by the peptide being limited to the gating properties of the ion channel. Dose dependency studies of GIRK activation by purified #beta##gamma# subunits of the G protein (G_#beta#_#gamma#) showed that DS6 block of GIRK channels is not the result of competition of the peptide with functional GIRK channels for the available G_#beta#_#gamma#; it is suggested that the blocking effect exerted by the peptide could be a quite intricate one and endow the corresponding (to DS6 peptide) aminoacids domain from the C-terminal of the GIRK1 subunit with major functional roles in the ruling the modulation of the GIRK1/GIRK5 kinetic. Hypothetically, this domain (similar in primary structure to the DS6 peptide) would possibly constitute itself as part of a blocking gate of the channel which keeps it closed prior interaction with G_#beta#_#gamma# subunits.
Cuvinte cheie: ion channels, patch-clamp, G-proteins, inward-rectification