Bile Acids Stimulate ATP Hydrolysis in the Purified Cholesterol Transporter ABCG5/G8

Abstract

ABCG5 and ABCG8 are half-size ABC transporters that function as heterodimers (ABCG5/G8) to reduce sterol absorption in the intestines and increase sterol excretion from the liver. Previous studies demonstrated that bile acids increased ABCG5/G8 specific cholesterol efflux in cell models. In this study we tested the effects of bile acids on ATP hydrolysis in Pichia pastoris purified ABCG5/G8 and found that they stimulated hydrolysis approximately 20-fold in wild-type ABCG5/G8 but not in a hydrolysis-deficient mutant. Nonconjugated cholate supported the highest ATPase activity in ABCG5/G8 (256 +/- 9 nmol min(-1) mg(-1)). ATP hydrolysis was also stimulated by other conjugated bile acids and a mixture of bile acids resembling human bile with activities ranging from 129 +/- 4 to 147 +/- 14 nmol min(-1) mg(-1). The kinetic parameters, inhibitor profiles, and lipid requirements of bile acid stimulated ATP hydrolysis were characterized. Cholate-stimulated ATP hydrolysis was maximal at concentrations of >or=10 mM MgATP and had a relatively high K(M) (MgATP) of approximately 1 mM. Orthovanadate, BeFx, and AlFx effectively inhibited ABCG5/G8 at concentrations of 1 mM. Various lipid mixtures supported bile acid-stimulated ATP hydrolysis, which increased when cholesterol was present. The data demonstrate that bile acids together with lipids and cholesterol increase ATP hydrolysis in purified ABCG5/G8. Bile acids may promote an active conformation of purified ABCG5/G8 either by global stabilization of the transporter or by binding to a specific site on ABCG5/G8.

Cholesterol lowering and inhibition of sterol absorption by Lactobacillus reuteri NCIMB 30242: a randomized controlled trial

“…43 Furthermore, it has been shown that overexpression of ABCG5 and ABCG8 in transgenic mice limits sterol absorption and promotes neutral sterol excretion 44 and that deconjugated bile acids preferentially increase ATPase activity of ABCG5/G8 transporters found on the apical membranes of enterocytes and hepatocytes, limiting the accumulation of cholesterol by transporting it into the intestinal lumen and bile. 45 The present results indicate that increased intraluminal BSH activity, in response to L. reuteri NCIMB 30242 supplementation, leads to an increase in deconjugated bile acids, a reduction in non-cholesterol sterol absorption and serum cholesterol, which is consistent with much of these hypotheses. The possibility, however, that the observed changes in sterol absorption may be a result of Niemann-Pick C1-like 1 transporter inhibition or a combination of cholesterol-reducing mechanisms should be considered.…”

ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin

“…ABCG1 did not show any ATPase activity without reconstitution, indicating that the lipid bilayer environment is crucial for the function of ABCG1. The ATPase activity (about 150 nmol/ min/mg) of ABCG1 was lower than that of ABCA1, MDR1, and TAP (450, 1700, and 1920 nmol/min/mg, respectively) ( 13,22,23 ) but was similar to that of ABCA4 and ABCG5/G8 (92 and 260 nmol/min/mg, respectively) ( 24,25 ). Thus, the ATPase activity of ABCG1 is high enough to function as an active transporter.…”

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3

“…In ABC transporters, binding of the substrate to the TMD typically stimulates ATP hydrolysis at the NBDs, and the energy from the hydrolysis is used to translocate the substrate across the membrane (52). The modulation of ATP hydrolysis upon substrate binding and translocation has been reported for several ABC transporters and is frequently used as a readout of transporter function and activity (36,40,44,53,54). However, no specific ATPase activity of MDR3 has been reported so far (16,17).…”

“…These findings confirm that MDR3 NBDs are able to accomplish substrate-stimulated ATP hydrolysis in the detergent-solubilized state. Despite the high degree of amino acid sequence identity between MDR1 and MDR3 (Ͼ85% homology to human MDR1 and 80% to mouse Mdr1a (previously called mouse MDR3)), they exhibited different maximal ATPase activities in the presence of the transport substrate but similar and relatively high K m values for ATP (MDR3, K m ϭ 1.90 Ϯ 0.27 mM; MDR1, K m ϭ 1.5 mM (44)), which were in a range typical for ABC transporters (36,40,44,57). Because PC lipids are present in high concentrations in the plasma membrane, Ishigami et al (17) suggested that MDR3 is a low affinity transporter optimized for PC translocation.…”