Sg. Huang et M. Klingenberg, CHLORIDE CHANNEL PROPERTIES OF THE UNCOUPLING PROTEIN FROM BROWN ADIPOSE-TISSUE MITOCHONDRIA - A PATCH-CLAMP STUDY, Biochemistry, 35(51), 1996, pp. 16806-16814
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.