Ion channel properties of the reconstituted chloroplast triose phosphate/phosphate translocator.

Schwarz, Martin; Gross, Armin; Steinkamp, Thomas; Flügge, Ulf‐Ingo; Wagner, Richard

doi:10.1016/s0021-9258(18)43905-1

Cited by 58 publications

(17 citation statements)

References 39 publications

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“…The I-V plot, shown in the inset to Patches excised from giant liposomes containing no UCP were electrically silent at V hold between -160 and 160 mV (n ) 4; not shown). This finding is consistent with previous reports (Criado & Keller, 1987;Keller et al, 1988;Schmid et al, 1988Schmid et al, , 1989Riquelme et al, 1990;Schwarz et al, 1994). Our standard procedure employs a lipid/protein ratio of 1400 (wt/wt) for the reconstitution.…”

Section: Resultssupporting

confidence: 93%

“…In line with the previously published procedure for generating giant liposomes for patch-clamp measurements (Schwarz et al, 1994), we used a buffer containing 2 mM Mg 2+ and in the pipet additionally 1 mM Ca 2+ . We investigated whether these ions can modulate the channel properties.…”

Section: Resultsmentioning

confidence: 99%

“…Very recently the isolated and reconstituted ADP/ ATP carrier was shown to exhibit channel behavior with high conductance and low specificity (Brustovetsky & Klingenberg, 1996). Also, ionic channel properties were observed in reconstituted chloroplast phosphate/triosephosphate carrier (Schwarz et al, 1994). In less related cases, transporters such as Ca 2+ -ATPase (Shamoo & MacLennan, 1975) and Na + ,K + -ATPase (Last et al, 1983;Mironova et al, 1986) were shown to behave like ion channels.…”

Section: Discussionmentioning

confidence: 99%

“…Reconstitution of UCP into Giant Liposomes. A freeze/ thawing procedure was employed to prepare the proteoliposomes (Schwarz et al, 1994) as follows. A stock suspension of azolectin was prepared by sonicating acetone-washed azolectin (50 mg/mL) on a Branson sonifier for 10 min in a buffer consisting of 5 mM Mes, 5 mM Tris, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

“…In view of a recent demonstration of channel-type Clconductance in the phosphate/triosephosphate exchanger from chloroplasts (Schwarz et al, 1994) and our own interest in exploring other methods to examine the transport modes of UCP, we embarked on patch-clamp studies of the UCP. Since the UCP is located in the inner mitochondrial membrane, it is difficult to obtain patches on the small mitoplast, let alone the presence of other channels which might interfere in the inner membrane Klitsch & Siemen, 1991).…”

mentioning

confidence: 99%

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

Huang¹,

Klingenberg²

1996

Biochemistry

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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.

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