Chloride Channel Properties of the Uncoupling Protein from Brown Adipose Tissue Mitochondria:  A Patch-Clamp Study

Abstract

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.

The Mitochondrial Uncoupling Protein UCP1: A Gated Pore

“…Thus, modi cation of one type of residues led to inhibition of transport while modi cation of another type led to a major loss of transport speci city, and it seemed to convert the carrier into a pore-like state (11)(12)(13). These channel-like properties were later con rmed by patch-clamp experiments that described conductances of up to 600 pS in the AAC (14) and lower but signi cant levels in the PiC (15) and UCP1 (16). All these results have been taken as an indication of the existence of at least two different functional domains in these carriers: one responsible for the carrier properties and another that confers channel-like properties (17)(18)(19).…”

Mitochondrial Ion Channels

“…A 75pS anion channel was recorded in giant liposomes reconstituted with UCP1 (88), and reconstitution of the individual transmembrane domains of human UCP2 showed that all six transmembrane peptides form helical conformations in lipid model membranes. Only the second transmembrane peptide exhibited voltage-dependent anion channel behavior (89).…”

Identification of a Functioning Mitochondrial Uncoupling Protein 1 in Thymus

“…UCP 1 transcripts in BAT have been determined by using Northern blot analysis, and the UCP 1 protein has usually been detected by using polyclonal antibodies specific for the fulllength protein. In addition, UCP 1 abundance in BAT (12)(13)(14) and the degree of masking (binding sites already occupied) of the purine nucleotide-binding sites (15) have been measured by binding of labeled GDP to mitochondria. To date, UCP 1 protein has not been found in any tissue other than BAT, although it has been sought in liver, heart, epididymal white adipose tissue, parametrial white adipose tissue, and thigh muscle (4,16); and a previous study, using Northern blot analysis, failed to detect UCP 1 transcripts in thymus (17).…”