Alkylsulfonates as probes of uncoupling protein transport mechanism - Ion pair transport demonstrates that direct H+ translocation by UCP1 is not necessary for uncoupling

The mechanism of fatty acid-dependent uncoupling by mitochondrial uncouplin g proteins (UCP) is still in debate. We have hypothesized that the anionic fatty acid head group is translocated by UCP, and the proton is transported electroneutrally in the bilayer by flip-flop of the protonated fatty acid. Alkylsulfonates are useful as probes of the UCP transport mechanism. They are analogues of fatty acids, and they are transported by UCP1, UCP2, and U CP3. We show that undecanesulfonate and laurate are mutually competitive in hibitors, supporting the hypothesis that fatty acid anion is transported by UCP1. Alkylsulfonates cannot be protonated because of their low pKa, conse quently, they cannot catalyze electroneutral proton transport in the bilaye r and cannot support uncoupling by UCP. We report for the first time that p ropranolol forms permeant ion pairs with the alkylsulfonates, thereby remov ing this restriction. Because a proton is transported with the neutral ion pair, the sulfonate is able to deliver protons across the bilayer, behaving as if it were a fatty acid. When ion pair transport is combined with UCP1, we now observe electrophoretic proton transport and uncoupling of brown ad ipose tissue mitochondria. These experiments confirm that the proton transp ort of UCP-mediated uncoupling takes place in the lipid bilayer and not via UCP itself. Thus, UCP1, like other members of its gene family, translocate s anions and does not translocate protons.