Chloride channel properties of the uncoupling protein from brown adipose tissue mitochondria: A patch-clamp study

The uncoupling protein (UCP) from brown adipose tissue mitochondria possesses H+ and Cl- transport activities [reviewed in Klingenberg, M. (1990) Trends Biochem. Sci. 15, 108-117]. Being a member of a mitochondrial carrier family, the transport of H+ and Cl- is carrier-like, i.e., much slower as compared to channels. Here we report that UCP reconstituted into giant liposomes displays stable chloride channel properties under patch-clamp conditions, The transport inhibitors (GTP, GDP, ATP, and ADP) also inhibit this channel in a reversible way, showing that the channel activity is associated with UCP. The slightly inward-rectifying chloride channel has a unit conductance of similar to 75 pS in symmetrical 100 mM KCl and closes at high positive potentials on the matrix side of UCP. Channel gatings switch from slow open-closure transitions to fast flickerings as the holding potential increases over +60 mV, Substitution experiments reveal a strong discrimination against cations [P(Cl-)/P(K+) approximate to 17] and a permeability ratio order of Cl- > Br- > F- > SCN- > I- > NO3- > SO42- > HPO42- > gluconate. Nucleotide inhibition studies indicate that 70% UCP molecules had its matrix side oriented outside in the giant liposomes. Fatty acids, pH, divalent cations (Ca2+ and Mg2+), and mersalyl do not influence these Cl- currents. The Cl- channel can be blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) from the matrix side of UCP. The data are consistent with a dimer consisting of two monomeric 75-pS Cl- channels or with a monomeric 150-pS channel having a 50% subconductance state. The channel current increases with Cl- concentration showing a typical saturation curve with K-m approximate to 63 mM and g(max) approximate to 120 pS (100 mM KCl in the piper). The Cl- conductance measured under these conditions is 6 orders of magnitude higher than the Cl- transport activity reported earlier, suggesting that the UCP has the potential of behaving as an anion channel.