PT  - JOURNAL ARTICLE
AU  - Brignac-Huber, Lauren M.
AU  - Reed, James R.
AU  - Eyer, Marilyn K.
AU  - Backes, Wayne L.
TI  - Relationship between CYP1A2 Localization and Lipid Microdomain Formation as a Function of Lipid Composition
AID  - 10.1124/dmd.113.053611
DP  - 2013 Nov 01
TA  - Drug Metabolism and Disposition
PG  - 1896--1905
VI  - 41
IP  - 11
4099  - http://dmd.aspetjournals.org/content/41/11/1896.short
4100  - http://dmd.aspetjournals.org/content/41/11/1896.full
SO  - Drug Metab Dispos2013 Nov 01; 41
AB  - Cytochrome P450 (P450) function requires the interaction of P450 and NADPH-cytochrome P450 reductase (CPR) in membranes, and is frequently studied using reconstituted systems composed solely of phosphatidylcholine. There is increasing evidence that other endoplasmic reticulum (ER) lipids can affect P450 structure, activity, and interactions with CPR. Some of these lipid effects have been attributed to the formation of organized liquid-ordered (lo) domains. The goal of this study was to determine if lo domains were formed in P450 reconstituted systems mimicking the ER membrane. CYP1A2, when incorporated in “ER-like” lipid vesicles, displayed detergent insolubility after treatment with Brij 98 and centrifugation in a sucrose gradient. Lipid probes were employed to identify domain formation in both ER-like vesicles and model membranes known to form lo domains. Changes in fluorescence resonance energy transfer (FRET) using an established donor/acceptor FRET pair in both ER-like and model lo-forming systems demonstrated the coexistence of lo- and liquid-disordered domains as a function of cholesterol and sphingomyelin content. Similarly, 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan), a probe that reports on membrane organization, showed that cholesterol and sphingomyelin increased membrane order. Finally, brominated-phosphatidylcholine allowed for monitoring of the location of both CPR and CYP1A2 within the lo regions of ER-like systems. Taken together, the results demonstrate that ER-like vesicles generate microdomains, and both CYP1A2 and CPR predominantly localize into lo membrane regions. Probe fluorescent responses suggest that lipid microdomains form in these vesicles whether or not enzymes are included in the reconstituted systems. Thus, it does not appear that the proteins are critical for stabilizing lo domains.