26 research outputs found
A single active catalytic site is sufficient to promote transport in P-glycoprotein
P-glycoprotein (Pgp) is an ABC transporter responsible for
the ATP-dependent efflux of chemotherapeutic compounds from
multidrug resistant cancer cells. Better understanding of the
molecular mechanism of Pgp-mediated transport could promote
rational drug design to circumvent multidrug resistance. By
measuring drug binding affinity and reactivity to a
conformation-sensitive antibody we show here that nucleotide
binding drives Pgp from a high to a low substrate-affinity
state and this switch coincides with the flip from the
inward- to the outward-facing conformation. Furthermore, the
outward-facing conformation survives ATP hydrolysis: the
post-hydrolytic complex is stabilized by vanadate, and the
slow recovery from this state requires two functional
catalytic sites. The catalytically inactive double Walker A
mutant is stabilized in a high substrate affinity inward-open
conformation, but mutants with one intact catalytic center
preserve their ability to hydrolyze ATP and to promote drug
transport, suggesting that the two catalytic sites are
randomly recruited for ATP hydrolysis
Fitting the Elementary Rate Constants of the P-gp Transporter Network in the hMDR1-MDCK Confluent Cell Monolayer Using a Particle Swarm Algorithm
P-glycoprotein, a human multidrug resistance transporter, has been extensively studied due to its importance to human health and disease. In order to understand transport kinetics via P-gp, confluent cell monolayers overexpressing P-gp are widely used. The purpose of this study is to obtain the mass action elementary rate constants for P-gp's transport and to functionally characterize members of P-gp's network, i.e., other transporters that transport P-gp substrates in hMDR1-MDCKII confluent cell monolayers and are essential to the net substrate flux. Transport of a range of concentrations of amprenavir, loperamide, quinidine and digoxin across the confluent monolayer of cells was measured in both directions, apical to basolateral and basolateral to apical. We developed a global optimization algorithm using the Particle Swarm method that can simultaneously fit all datasets to yield accurate and exhaustive fits of these elementary rate constants. The statistical sensitivity of the fitted values was determined by using 24 identical replicate fits, yielding simple averages and standard deviations for all of the kinetic parameters, including the efflux active P-gp surface density. Digoxin required additional basolateral and apical transporters, while loperamide required just a basolateral tranporter. The data were better fit by assuming bidirectional transporters, rather than active importers, suggesting that they are not MRP or active OATP transporters. The P-gp efflux rate constants for quinidine and digoxin were about 3-fold smaller than reported ATP hydrolysis rate constants from P-gp proteoliposomes. This suggests a roughly 3∶1 stoichiometry between ATP hydrolysis and P-gp transport for these two drugs. The fitted values of the elementary rate constants for these P-gp substrates support the hypotheses that the selective pressures on P-gp are to maintain a broad substrate range and to keep xenobiotics out of the cytosol, but not out of the apical membrane
Notch controls embryonic Schwann cell differentiation, postnatal myelination and adult plasticity
Notch signaling is central to vertebrate development, and analysis of Notch has provided important insights into pathogenetic mechanisms in the CNS and many other tissues. However, surprisingly little is known about the role of Notch in the development and pathology of Schwann cells and peripheral nerves. Using transgenic mice and cell cultures, we found that Notch has complex and extensive regulatory functions in Schwann cells. Notch promoted the generation of Schwann cells from Schwann cell precursors and regulated the size of the Schwann cell pool by controlling proliferation. Notch inhibited myelination, establishing that myelination is subject to negative transcriptional regulation that opposes forward drives such as Krox20. Notably, in the adult, Notch dysregulation resulted in demyelination; this finding identifies a signaling pathway that induces myelin breakdown in vivo. These findings are relevant for understanding the molecular mechanisms that control Schwann cell plasticity and underlie nerve pathology, including demyelinating neuropathies and tumorigenesi