KChannelDB: Extraction of mutation data from the literature

This data was extracted from Medline abstracts and full texts (when available) in an automated manner.

The table below describes the selected point mutation and provides links to other documents. The sentence(s) where the point mutation S329A was found are listed after the table.

The mutated residues are also indicated in the family sequence alignments and hyperlinked to the corresponding mutation pages.

Point mutation S329A in IRK15_RAT

Relevant sentences:

S329A

• 304 , Issue 3 , 924-930 , March 2003 Serine 329 of the µ-Opioid Receptor Interacts Differently with Agonists Joost Pil and Jan Tytgat Laboratory of Toxicology , Faculty of Pharmaceutical Sciences , University of Leuven , Leuven , Belgium Abstract (image)Top (image)Abstract (image)Introduction (image)Materials and Methods (image)Results (image)Discussion (image)References To investigate the effect of the hydrophilic Ser amino acid in position 329 of the human µ-opioid receptor (hMORwt) on the potency of various agonists , we mutated this residue to Ala (hMORS329A)

• Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric G-protein-coupled inwardly rectifying potassium channel (GIRK1 / GIRK2) , either the wild-type hMOR or the mutated receptor (hMORS329A) was functionally coexpressed with GIRK1 and GIRK2 channels together with a regulator of G-protein signaling (RGS4) in Xenopus laevis oocytes

• The potency of the peptide agonist [D-Ala2 , N-MePhe4 , Gly5-ol]-enkephalin (DAMGO) decreased as measured via hMORS329A , whereas the potency of nonpeptide agonists like morphine , fentanyl , and beta -hydroxyfentanyl (R004333) increased via the mutated receptor

• This experimentally created model (Ulens et al. , 2000b(image) ) provides a defined population of functionally active opioid receptor (hMORwt or hMORS329A) and an excellent tool for measuring the efficacy and potency of a ligand for a certain receptor (Pil and Tytgat , 2001(image) )

• For in vitro transcription , the mutant hMORS329A / pGEMHE was linearized with NheI

• Oocytes were coinjected with 0.5 ng 50 nl -1 GIRK1 , 0.5 ng 50 nl -1 GIRK2 , and 10 ng 50 nl -1 RGS4 cRNA , with the addition of 10 ng 50 nl -1 of either hMOR or hMORS329A cRNA

• In our study , we examined the potency of DAMGO , morphine , fentanyl , and beta -hydroxyfentanyl (Fig. 2 ) on hMORwt and on the mutant receptor hMORS329A

• Figure 3 shows representative current traces of agonist-gated currents evoked from oocytes expressing either hMORwt (Fig. 3 A) or hMORS329A (Fig. 3 B) by DAMGO

• laevis oocytes coexpressing GIRK1 / GIRK2 channels and RGS4 with hMORwt (A) or hMORS329A (B)

• laevis oocytes coexpressing GIRK1 / GIRK2 and RGS4 with hMORwt (black-diamond ) or hMORS329A (black-square)

• DAMGO , a µ-opioid-selective peptidic agonist was more than 4 times less potent via the mutant hMORS329A receptor (EC50 value: 111.1 ± 22.3 nM) compared with the wild-type receptor (EC50 value: 25.6 ± 5.0 nM)

• On the wild-type receptor , hydroxyfentanyl (EC50 value: 38.1 ± 2.1 nM) was more potent than fentanyl (EC50 value: 64.9 ± 7.8 nM) , whereas fentanyl activates GIRK1 / GIRK2 channels with a significantly higher potency than hydroxyfentanyl on the hMORS329A mutant receptor (EC50 values: 9.3 ± 0.3 nM for fentanyl and 24.5 ± 0.5 nM for hydroxyfentanyl)

• The potency of morphine was increased 5-fold by mutating the wild-type hMOR to hMORS329A

• We conclude that additional hydrophobic contacts between Ala329 in the mutant receptor and the phenyl ring of morphine can explain the increase in potency of morphine via the hMORS329A receptor (Fig. 7 A)

• The potency of fentanyl was increased 7-fold by mutating the wild-type receptor to hMORS329A

• The mutation of Ser329 to a lipophilic Ala329 can create a more favorable vicinity for the N-phenethyl group of fentanyl to interact deep within the binding cavity , thereby clarifying the increased potency of fentanyl on hMORS329A

• Surprisingly , a rather small , albeit significantly , increase in potency was observed with beta -hydroxyfentanyl on HMORS329A compared with HMORwt

• Remarkably however , on the hMORS329A receptor fentanyl is more potent compared with beta -hydroxyfentanyl

Ser329

• Our results are indicative for the existence of hydrophilic interactions between Ser329 and DAMGO , thereby decreasing the potency of DAMGO via the mutated receptor , whereas hydrophobic interactions between the mutated receptor and the N-phenylethyl of morphine and fentanyl can explain the increased potency

• We conclude that the hydroxyl group of Ser329 is not involved in the formation of a hydrogen bond with the beta -hydroxy group of fentanyl and that mutation of this residue to alanine caused dual effects depending on the nature of the ligand

• The circle indicates the position of Ser329

• The goal of this work was to examine the importance of Ser329 in the human µ-opioid receptor , thereby contributing to define the orientation of the phenethyl ring within the receptor cavity

• For clarity , the Ser329 corresponds to the Ser331 in the clone of Subramanian et al. (2000)(image)

• Therefore , we mutated this Ser329 to Ala329 and determined the EC50 values for GIRK1 / GIRK2 channel activation through consecutive activation of wild-type and mutant opioid receptors coupled to G-proteins and expressed in Xenopus laevis oocytes

• Ser329 in hMOR was mutated to Ala329 using the Quickchange site-directed mutagenesis kit (Stratagene , La Jolla , CA)

• The Ser329 mutation we made is located in the middle of these (Tyr326 and Asp332) mutations

• The mutation of Ser329 to Ala329 caused a more than 4-fold decrease in potency for DAMGO , suggesting the existence of hydrophilic interactions between DAMGO and the serine residue

• The carboxyl terminal hydroxyl group is a possible candidate to form a hydrogen bond with Ser329

• According to Pogozheva et al. (1998)(image) , Ser329 , together with Asn86 , Asp114 , Ser154 , Asn328 , and Asn332 , is a part of a highly conserved polar cluster

• Surprisingly , mutation of this Ser329 to Alanine , thereby disturbing this putative cluster , can enhance the potency of morphine toward the mutant receptor

• The side chains of Asp147 and Ser329 are represented in ball-and-stick form with oxygen , carbon , and nitrogen colored in red , black , and blue , respectively

• A , the quaternary nitrogen of morphine (shown in blue) interacts with Asp147 , whereas the phenyl ring lies close to Ser329

• B , the piperidine nitrogen of fentanyl (colored in blue) forms electrostatic interactions with Asp147 , whereas the N-phenethyl group of fentanyl extends within the binding cavity to Ser329

• These residues correspond to Asn328 , Ser329 , Asn332 , and Pro333 , respectively , in our human receptor

• The mutation of Ser329 to a lipophilic Ala329 can create a more favorable vicinity for the N-phenethyl group of fentanyl to interact deep within the binding cavity , thereby clarifying the increased potency of fentanyl on hMORS329A

• We expected a decreased potency on the assumption that Ser329 serves as a hydrogen-bonding partner with the beta -hydroxyl group of the hydroxyfentanyl derivatives , as suggested by Subramanian et al. (2000)(image)

• It is unlikely that Ser329 forms a hydrogen bond with this hydroxyl group , but an interaction with another part of the N-phenethyl groups remains possible as Ala329 exert a favorable influence

• In conclusion , the present studies demonstrate the dual role of Ser329 in the potency of various agonists

F.Horn (kchanneldbcmbi.ru.nl), 17-Aug-2005