Transport kinetics of uncoupling proteins. Analysis of UCP1 reconstituted in planar lipid bilayers.
According to alternative hypotheses, mitochondrial uncoupling protein 1 (UCP1) is either a proton channel ("buffering model") or a fatty acid anion carrier ("fatty acid cycling"). Transport across the proton channel along a chain of hydrogen bonds (Grotthus mechanism) may include fatty acid carboxyl groups or occur in the absence of fatty acids. In this work, we demonstrate that planar bilayers reconstituted with UCP1 exhibit an increase in membrane conductivity exclusively in the presence of fatty acids. Hence, we can exclude the hypothesis considering a preexisting H+ channel in UCP1, which does not require fatty acid for function. The augmented conductivity is nearly completely blocked by ATP. Direct application of transmembrane voltage and precise current measurements allowed determination of ATP-sensitive conductances at 0 and 150 mV as 11.5 and 54.3 pS, respectively, by reconstituting nearly 3 x 10(5) copies of UCP1. The proton conductivity measurements carried out in presence of a pH gradient (0.4 units) allowed estimation of proton turnover numbers per UCP1 molecule. The observed transport rate of 14 s-1 is compatible both with carrier and channel nature of UCP1.
Urbánková
Eva
E
Department of Membrane Transport Biophysics, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Voltchenko
Anna
A
Pohl
Peter
P
Jezek
Petr
P
Pohl
Elena E
EE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D008647:Mesocricetus; D024101:Mitochondrial Proteins; D019301:Oleic Acid / Q000494:pharmacology; D021381:Protein Transport; D011510:Proteolipids / Q000378:metabolism; D011522:Protons; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D005227:Fatty Acids; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011510:Proteolipids; D011522:Protons; C000604371:UCP1 protein, human; D000071256:Uncoupling Protein 1; C020485:proteoliposomes; D019301:Oleic Acid; D000255:Adenosine Triphosphate