Mutational Analysis of the P-Glycoprotein First Intracellular Loop and Flanking Transmembrane Domains†

Biochemistry ◽  
1998 ◽  
Vol 37 (10) ◽  
pp. 3337-3350 ◽  
Author(s):  
Tony Kwan ◽  
Philippe Gros
Keyword(s):  
Mutational Analysis ◽  
Transmembrane Domains ◽  
Intracellular Loop ◽  
P Glycoprotein

scholarly journals Combination of Suboptimal Doses of Inhibitors Targeting Different Domains of LtrMDR1 Efficiently Overcomes Resistance of Leishmania spp. to Miltefosine by Inhibiting Drug Efflux

2006 ◽  
Vol 50 (9) ◽  
pp. 3102-3110 ◽  
Author(s):  
José M. Pérez-Victoria ◽  
Fernando Cortés-Selva ◽  
Adriana Parodi-Talice ◽  
Boris I. Bavchvarov ◽  
F. Javier Pérez-Victoria ◽  
...  
Keyword(s):  
Active Drug ◽  
Transmembrane Domains ◽  
Drug Efflux ◽  
Binding Domains ◽  
Mammalian Cell Lines ◽  
Structure Activity ◽  
P Glycoprotein ◽  
Leishmania Spp ◽  
Orally Active ◽  
Glycoprotein Inhibitors

ABSTRACT Miltefosine (hexadecylphosphocholine) is the first orally active drug approved for the treatment of leishmaniasis. We have previously shown the involvement of LtrMDR1, a P-glycoprotein-like transporter belonging to the ATP-binding cassette superfamily, in miltefosine resistance in Leishmania. Here we show that overexpression of LtrMDR1 increases miltefosine efflux, leading to a decrease in drug accumulation in the parasites. Although LtrMDR1 modulation might be an efficient way to overcome this resistance, a main drawback associated with the use of P-glycoprotein inhibitors is related to their intrinsic toxicity. In order to diminish possible side effects, we have combined suboptimal doses of modulators targeting both the cytosolic and transmembrane domains of LtrMDR1. Preliminary structure-activity relationships have allowed us to design a new and potent flavonoid derivative with high affinity for the cytosolic nucleotide-binding domains. As modulators directed to the transmembrane domains, we have selected one of the most potent dihydro-β-agarofuran sesquiterpenes described, and we have also studied the effects of two of the most promising, latest-developed modulators of human P-glycoprotein, zosuquidar (LY335979) and elacridar (GF120918). The results show that this combinatorial strategy efficiently overcomes P-glycoprotein-mediated parasite miltefosine resistance by increasing intracellular miltefosine accumulation without any side effect in the parental, sensitive, Leishmania line and in different mammalian cell lines.

scholarly journals Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating

2018 ◽  
Vol 115 (22) ◽  
pp. E5193-E5202 ◽  
Author(s):  
Priscilla S.-W. Yeung ◽  
Megumi Yamashita ◽  
Christopher E. Ing ◽  
Régis Pomès ◽  
Douglas M. Freymann ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mutational Analysis ◽  
Channel Gating ◽  
Functional Anatomy ◽  
Transmembrane Domains ◽  
Hydrophobic Residues ◽  
Binding Domains ◽  
Allosteric Interactions ◽  
Crac Channel ◽  
Dynamics Simulations

Store-operated Orai1 channels are activated through a unique inside-out mechanism involving binding of the endoplasmic reticulum Ca2+ sensor STIM1 to cytoplasmic sites on Orai1. Although atomic-level details of Orai structure, including the pore and putative ligand binding domains, are resolved, how the gating signal is communicated to the pore and opens the gate is unknown. To address this issue, we used scanning mutagenesis to identify 15 residues in transmembrane domains (TMs) 1–4 whose perturbation activates Orai1 channels independently of STIM1. Cysteine accessibility analysis and molecular-dynamics simulations indicated that constitutive activation of the most robust variant, H134S, arises from a pore conformational change that opens a hydrophobic gate to augment pore hydration, similar to gating evoked by STIM1. Mutational analysis of this locus suggests that H134 acts as steric brake to stabilize the closed state of the channel. In addition, atomic packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set mediated by a cluster of interdigitating hydrophobic residues and another by alternative ridges of polar and hydrophobic residues. Perturbing these contacts via mutagenesis destabilizes STIM1-mediated Orai1 channel gating, indicating that these bridges between TM1 and the surrounding TM2/3 ring are critical for conveying the gating signal to the pore. These findings help develop a framework for understanding the global conformational changes and allosteric interactions between topologically distinct domains that are essential for activation of Orai1 channels.

Mutational analysis of predicted intracellular loop domains of human motilin receptor

2008 ◽  
Vol 294 (2) ◽  
pp. G460-G466 ◽  
Author(s):  
Hitoo Tokunaga ◽  
Bunzo Matsuura ◽  
Maoqing Dong ◽  
Laurence J. Miller ◽  
Teruhisa Ueda ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
G Protein ◽  
Mutational Analysis ◽  
Negative Impact ◽  
Intracellular Loop ◽  
Gastrointestinal Motor ◽  
Intracellular Loops ◽  
Motilin Receptor ◽  
Loop Domains ◽  
G Protein Coupled

Motilin is an important endogenous regulator of gastrointestinal motor function, mediated by the class I G protein-coupled motilin receptor. Motilin and erythromycin, two chemically distinct full agonists of the motilin receptor, are known to bind to distinct regions of this receptor, based on previous systematic mutagenesis of extracellular regions that dissociated the effects on these two agents. In the present work, we examined the predicted intracellular loop regions of this receptor for effects on motilin- and erythromycin-stimulated activity. We prepared motilin receptor constructs that included sequential deletions throughout the predicted first, second, and third intracellular loops, as well as replacing the residues in key regions with alanine, phenylalanine, or histidine. Each construct was transiently expressed in COS cells and characterized for motilin- and erythromycin-stimulated intracellular calcium responses and for motilin binding. Deletions of receptor residues 63–66, 135–137, and 296–301 each resulted in substantial loss of intracellular calcium responses to stimulation by both motilin and erythromycin. Constructs with mutations of residues Tyr66, Arg136, and Val299 were responsible for the negative impact on biological activity stimulated by both agonists. These data suggest that action by different chemical classes of agonists that are known to interact with distinct regions of the motilin receptor likely yield a common activation state of the cytosolic face of this receptor that is responsible for interaction with its G protein. The identification of functionally important residues in the predicted cytosolic face provides strong candidates for playing roles in receptor-G protein interaction.

scholarly journals Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux

2004 ◽  
Vol 11 (9) ◽  
pp. 1028-1037 ◽  
Author(s):  
K M Tainton ◽  
M J Smyth ◽  
J T Jackson ◽  
J E Tanner ◽  
L Cerruti ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Drug Efflux ◽  
Caspase Activation ◽  
P Glycoprotein
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