Single anion channels reconstituted from cardiac mitoplasts

Hayman, K. A.; Spurway, Tracey D.; Ashley, Richard H.

doi:10.1007/bf02505762

Cited by 38 publications

(25 citation statements)

References 37 publications

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“…The preparation of sheep heart mitochondria (Toth, FergusonMiller & Suelter, 1986) and inner membrane (mitoplast membrane) vesicles (Chan, Greenwalt & Pedersen, 1970;Williams & Pedersen, 1986), together with the techniques used for planar bilayer reconstitution, and all the materials used, were the same as those described in detail in the preceding paper (Hayman et al, 1993). The cis bilayer chamber continued to be voltageclamped with respect to the trans chamber, which was grounded, and all potentials are reported as cis-trans.…”

Section: Methodsmentioning

confidence: 99%

“…In this report, the "low-conductance" anion channels identified in the previous paper (Hayman et al, 1993) were studied in detail. Interestingly, they were found to insert asymmetrically into the bilayer such that one face was located only about 1 nm from the membrane surface (as inferred from the effect of membrane surface potential on ion permeation), and while they also exhibited gating behavior consistent with a multibarrelled channel containing four functionally coupled pores, the allosteric subunit interactions involved appear to have been quite complicated.…”

Section: Introductionmentioning

confidence: 95%

“…The preceding paper (Hayman, Spurway & Ashley, 1993) described the incorporation into planar lipid bilayers of anion channels from sheep cardiac mitoplast (inner mitochondrial membrane vesicle) preparations. The channels were particularly rich in subconductance states between the predominant (in this case, the 100%) open state, and the shut state.…”

Section: Introductionmentioning

confidence: 99%

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Structural features of a multisubstate cardiac mitoplast anion channel: Inferences from single channel recording

Hayman

Ashley

1993

J. Membrain Biol.

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Ion channels from sheep cardiac mitoplast (inverted inner mitochondrial membrane vesicle) preparations were incorporated into voltage-clamped planar lipid bilayers. A low-conductance anion channel (approximately 40 or approximately 85 pS in symmetric 300 or 550 mM choline Cl, respectively), characterized by the presence of two well-defined substates, at approximately 25 and approximately 50% of the fully open level, was studied in detail. The substate behavior was consistent with a multibarelled channel containing four functionally coupled pores. At negative (cis-trans) membrane potentials, the putative protomers appeared to gate with substantial positive cooperativity, accounting for the apparent absence of a approximately 75% sublevel. At positive holding potentials, allosteric promoter interactions were more complicated, and the channel complex could be modeled as a dimer of dimers. The protochannels in one dimer ("dimer A") appeared to open independently of each other, and with a relatively high probability, while the monomers comprising the second dimer ("dimer B") were functionally coupled, could only open if both protomers in dimer A were open, and closed as soon as one of the monomers in dimer A shut. The channels also displayed Ca(2+)-(and Mg(2+)-) sensitive rectification related to bilayer lipid surface charge. By assuming that Ca2+ acted solely by screening surface charge, the membrane surface potential profile was used as a "microscopic ruler" to place one month of the channel within 10-11 A of the bilayer surface.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

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Structural features of a multisubstate cardiac mitoplast anion channel: Inferences from single channel recording

Hayman

Ashley

1993

J. Membrain Biol.

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“…One or more anion channels have been observed in the inner mitochondrial membrane using the electrophysiological techniques of patch-clamping (9 -11) and reconstitution into planar lipid bilayers (12,13). Whether any of these channels represent IMAC is still uncertain; however, many of the properties of IMAC in intact mitochondria can be explained by a simple channel model in which H ϩ and Mg 2ϩ decrease the open probability of IMAC by binding to the closed state.…”

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confidence: 99%

Temperature Dependence of the Mitochondrial Inner Membrane Anion Channel

Hinch

Davatol-Hag

et al. 1996

Journal of Biological Chemistry

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In this paper, we investigate the temperature and pH dependence of the mitochondrial inner membrane anion channel (IMAC) that is believed to be involved in mitochondrial volume homeostasis. At pH 7.4, the flux of malonate is highly temperature-dependent with rates increasing from 1 nmol/min⅐mg at 5°C to 1900 nmol/ min⅐mg at 45°C. The Arrhenius plot is nonlinear with the activation energy increasing from 21 kJ/mol (Q 10 ‫؍‬ 1.3) to 193 kJ/mol (Q 10 ‫؍‬ 13) as the temperature is decreased. This temperature dependence is unusual and not seen with solutes that are transported through the bilayer such as NH 4 OAc, malonamide, and KSCN (plus valinomycin) or even for cytochrome c oxidase-dependent uptake of potassium (plus valinomycin). The temperature dependence of IMAC is closely related to the inhibition of IMAC by protons. Thus, we find that the pIC 50 for protons decreases from 9.3 (Hill coefficient ‫؍‬ 1.0) at 5°C to 7.1 (Hill coefficient ‫؍‬ 2.5) at 45°C. This behavior is explained on the basis of a new kinetic model for IMAC in which the net open probability is not only modulated by the binding of three protons but also by temperature via effects on the open probability of the unprotonated channel and the pK of one of the inhibitory protonation sites.The mitochondrial inner membrane anion channel (IMAC) 1 mediates the transport of a wide variety of different anions and is believed to be involved in mitochondrial volume homeostasis. Its properties have been characterized at 25°C by flux studies in intact mitochondria, and many inhibitors have been identified (see Ref. 1 for a review). Recently, it has been shown that IMAC is blocked by DIDS (2) which is an inhibitor of many anion channels (3). The most physiologically important inhibitors of IMAC are protons and magnesium which inhibit from the matrix (inner) side of the inner membrane. At pH 7.4, the pIC 50 for Mg 2ϩ is about 40 M (4), and the pIC 50 for protons is about 7.8 (5); consequently, in freshly isolated mitochondria, which contain about 0.5 mM free Mg 2ϩ (6, 7), IMAC is almost inactive. Thus, IMAC is usually assayed in Mg 2ϩ -depleted mitochondria or under conditions that alkalinize the matrix (1). It has also been shown that N-ethylmaleimide and mercurials stimulate IMAC by increasing the IC 50 for protons and the IC 50 for Mg 2ϩ (8); moreover, the IC 50 for Mg 2ϩ is also found to increase with pH (4). These findings have led to the suggestion that other physiological factors may modulate the IC 50 values for these inhibitors (8). In the present paper, we show that temperature is one of these important factors.One or more anion channels have been observed in the inner mitochondrial membrane using the electrophysiological techniques of patch-clamping (9 -11) and reconstitution into planar lipid bilayers (12, 13). Whether any of these channels represent IMAC is still uncertain; however, many of the properties of IMAC in intact mitochondria can be explained by a simple channel model in which H ϩ and Mg 2ϩ decrease the open probability of IMAC by binding...

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“…The properties of IMAC and the 108-pS channel seemed to be similar [10]. Two distinct anion channels were identified in reconstituted cardiac mitoplasts [11]. Among the chloride channels observed in the inner mitochondrial membrane, there is only one -CLIC4 (also called mtCLIC or p64H1) -that has been cloned [12].…”

Section: Introductionmentioning

confidence: 99%

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Koszela-Piotrowska

Choma

Bednarczyk

et al. 2007

Cellular and Molecular Biology Letters

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Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70-130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 µM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50-100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.

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Single anion channels reconstituted from cardiac mitoplasts

Cited by 38 publications

References 37 publications

Structural features of a multisubstate cardiac mitoplast anion channel: Inferences from single channel recording

Structural features of a multisubstate cardiac mitoplast anion channel: Inferences from single channel recording

Temperature Dependence of the Mitochondrial Inner Membrane Anion Channel

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

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