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, 108-112]. 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 approximately 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+) approximately 17] and a permeability ratio order of Cl- > Br- > F- > SCN- > I- > NO3- > SO4(2-) > HPO4(2-) > 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 Km approximately 63 mM and gmax approximately 120 pS (100 mM KCl in the pipet). 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.
Huang
S G
SG
Institute of Physical Biochemistry, University of Munich, Germany. sghuang@pbm.med.uni-muenchen.de
Klingenberg
M
M
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004553:Electric Conductivity; D005227:Fatty Acids / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008634:Mersalyl / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D018408:Patch-Clamp Techniques; D011685:Purine Nucleotides / Q000494:pharmacology; D014475:Uncoupling Agents / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D018118:Chloride Channels; D005227:Fatty Acids; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011685:Purine Nucleotides; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; D008634:Mersalyl; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid