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 po ssesses H+ and Cl- transport activities [reviewed in Klingenberg, M. ( 1990) Trends Biochem. Sci. 15, 108-117]. Being a member of a mitochond rial carrier family, the transport of H+ and Cl- is carrier-like, i.e. , much slower as compared to channels. Here we report that UCP reconst ituted into giant liposomes displays stable chloride channel propertie s under patch-clamp conditions, The transport inhibitors (GTP, GDP, AT P, and ADP) also inhibit this channel in a reversible way, showing tha t the channel activity is associated with UCP. The slightly inward-rec tifying 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 tran sitions to fast flickerings as the holding potential increases over +6 0 mV, Substitution experiments reveal a strong discrimination against cations [P(Cl-)/P(K+) approximate to 17] and a permeability ratio orde r of Cl- > Br- > F- > SCN- > I- > NO3- > SO42- > HPO42- > gluconate. N ucleotide inhibition studies indicate that 70% UCP molecules had its m atrix side oriented outside in the giant liposomes. Fatty acids, pH, d ivalent cations (Ca2+ and Mg2+), and mersalyl do not influence these C l- currents. The Cl- channel can be blocked by 4,4'-diisothiocyanatost ilbene-2,2'-disulfonic acid (DIDS) from the matrix side of UCP. The da ta are consistent with a dimer consisting of two monomeric 75-pS Cl- c hannels 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) app roximate to 120 pS (100 mM KCl in the piper). The Cl- conductance meas ured under these conditions is 6 orders of magnitude higher than the C l- transport activity reported earlier, suggesting that the UCP has th e potential of behaving as an anion channel.