A Voltage‐dependent Chloride Conductance Channel From Torpedo Electroplax Membrane*

Miller, Christopher; White, Michael M.

doi:10.1111/j.1749-6632.1980.tb47197.x

Cited by 126 publications

(107 citation statements)

References 17 publications

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“…This membrane patch can be voltage clamped so that fluctuations in current due to the opening and closing of single Cl-channels are directly observed. The voltage-dependent gating and ion conduction properties ofCl-channels in these reconstituted membrane patches are identical to those reported for channels directly inserted into planar bilayers without previous exposure to detergent (15)(16)(17).MATERIAL AND METHODS Biochemical. Phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer) from bovine heart and brain, respectively, were obtained from Avanti Biochemicals.…”

mentioning

confidence: 82%

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Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.

Tank¹,

Miller²,

Webb³

1982

Proc. Natl. Acad. Sci. U.S.A.

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Small unilamellar vesicles formed from purified phospholids by detergent/dialysis methods may be enlarged to 30-,um diameter by freezing and thawing. Very-high-resistance seals were formed by applying a glass micropipette to the surface of these large liposomes, and single bilayer "patches" of membrane were isolated from the liposome surface while remaining sealed to the micropipette. The exogenous channel-forming peptides gramicidin and alamethicin induced characteristic single-channel fluctuation behavior in these excised patches held under voltageclamp conditions. Large liposomes were formed from the small unilamellar vesicles made from cholate extracts of Torpedo electroplax plasma membrane vesicles. Isolated patches formed from these reconstituted membranes displayed current fluctuations due to single voltage-gated Cl channels from non-innervated-face membranes; the properties of these Cl1 channels are identical to those observed in planar bilayer membranes after direct insertion from native membranes. This liposome-patch method combines the advantages ofmembrane protein incorporation into liposomes with high-resolution electrical recording methods and may provide a generally applicable approach to the study ofintegral membrane channel proteins after solubilization and reconstitution.In any complete biochemical study of a membrane transport system, it is necessary to solubilize the protein of interest, manipulate it in the solubilized state, and then reinsert it into a "reconstituted" membrane in which its solute-transport activity can be assayed. This "solubilization/reconstitution" approach has been applied to many classes of membrane transport systems (1, 2), with the single exception ofthe ion channel proteins of higher organisms. Only one ion channel protein, the nicotinic acetylcholine receptor (AcChoR), has been reconstituted (3-5). Ion channels have been resistant to classical reconstitution work because the small unilamellar vesicles (SUVs) used in such work present great difficulties in the measurement of channel-mediated ion fluxes; a "typical" SUV containing a channel of50-pS conductance would completely equilibrate its ion content in several milliseconds, far faster than the time resolution of the conventional vesicle flux assay (1, 6). A further problem is that many ion channels are controlled by membrane voltage, which cannot be reliably maintained constant during such assays. Methods do exist for inserting ion channels into planar bilayer membranes, which may be voltage clamped and subjected to electrical current assays (7-10), but these methods, unlike vesicle reconstitution methods, are not yet generally applicable; they work well for some channels but not for others (6).In this paper, we report that the solubilization/reconstitution method for inserting membrane proteins into SUVs can be combined with a high-resolution electrical assay technique to study a reconstituted integral membrane ion channel-the voltage-gated Cl-channel of Torpedo electroplax. The method is shown schemati...

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confidence: 82%

“…4. This channel has properties identifying it as the voltage-dependent Cl-channel from Torpedo electroplax, which has been studied in detail by fusion of native electroplax membranes with planar phospholipid bilayers (15)(16)(17). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.

Tank¹,

Miller²,

Webb³

1982

Proc. Natl. Acad. Sci. U.S.A.

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“…The first protein of the CLC family to be identified is ClC-0, a CLC present in the electric organ of torpedo fishes (Miller & White 1980). The cloning of the ClC-0 gene (Jentsch et al 1990) led to the identification of a wide family of related proteins whose members are present in many bacteria and in all eukaryotic organisms analysed up to now (Miller 2006).…”

Section: The Plant Clc Familymentioning

confidence: 99%

CLC-mediated anion transport in plant cells

Angeli

Monachello

Ephritikhine

et al. 2008

Phil. Trans. R. Soc. B

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Plants need nitrate for growth and store the major part of it in the central vacuole of cells from root and shoot tissues. Based on few studies on the two model plants Arabidopsis thaliana and rice, members of the large ChLoride Channel (CLC) family have been proposed to encode anion channels/transporters involved in nitrate homeostasis. Proteins from the Arabidopsis CLC family (AtClC, comprising seven members) are present in various membrane compartments including the vacuolar membrane (AtClCa), Golgi vesicles (AtClCd and AtClCf ) or chloroplast membranes (AtClCe). Through a combination of electrophysiological and genetic approaches, AtClCa was shown to function as a 2NO 3 K /1H C exchanger that is able to accumulate specifically nitrate into the vacuole, in agreement with the main phenotypic trait of knockout mutant plants that accumulate 50 per cent less nitrate than their wild-type counterparts. The set-up of a functional complementation assay relying on transient expression of AtClCa cDNA in the mutant background opens the way for studies on structure-function relationships of the AtClCa nitrate transporter. Such studies will reveal whether important structural determinants identified in bacterial or mammalian CLCs are also crucial for AtClCa transport activity and regulation.

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“…This channel has been characterized in detail, after insertion into planar lipid bilayers (1)(2)(3)(4)(5) or giant lipid vesicles (6, 7), by observation of current fluctuations under voltage-clamp conditions. At the single-channel level, an unusual gating behavior is observed: a "bursting" process in which the open channel displays three distinguishable "substates" (4,5).…”

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confidence: 99%

“…In the plasma membrane of the electric organ of Torpedo californica resides a Cl--specific channel whose function is to establish the electroplax cell as a low-internal-resistance battery that the fish may use as a source of electric current in stunning its prey (1)(2)(3)(4)(5). This channel has been characterized in detail, after insertion into planar lipid bilayers (1)(2)(3)(4)(5) or giant lipid vesicles (6, 7), by observation of current fluctuations under voltage-clamp conditions.…”

mentioning

confidence: 99%

Dimeric structure of single chloride channels from Torpedo electroplax.

Miller¹,

White²

1984

Proc. Natl. Acad. Sci. U.S.A.

239

157

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The inhibition by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) of Cl-channels from Torpedo electroplax incorporated in planar phospholipid bilayer membranes is studied. DIDS irreversibly and rapidly inhibits the macroscopic conductance of membranes containing many channels. At the single-channel level, the effect of DIDS is more complicated. The uninhibited single channel displays three "substates" of conductances 20, 10, and 0 pS. Short exposure (5-30 s) to 10 j&M DIDS converts this three-level active channel into a "conventional" channel of 10-pS conductance. Longer exposure eliminates all channel fluctuations. The results are taken as strong evidence that the ClF channel is constructed as a functional dimer of identical protein subunits.In the plasma membrane of the electric organ of Torpedo californica resides a Cl--specific channel whose function is to establish the electroplax cell as a low-internal-resistance battery that the fish may use as a source of electric current in stunning its prey (1-5). This channel has been characterized in detail, after insertion into planar lipid bilayers (1-5) or giant lipid vesicles (6, 7), by observation of current fluctuations under voltage-clamp conditions. At the single-channel level, an unusual gating behavior is observed: a "bursting" process in which the open channel displays three distinguishable "substates" (4,5). This open-channel substructure is uncommonly well-behaved and has led to a simple physical picture of the Cl-channel as a functional dimer. We have proposed that the three active substates result from the independent opening and closing of two identical Cl-diffusion pathways, or "protochannels" (4, 5). In this scheme, the dimeric channel complex may exist with both protochannels simultaneously open, with one open and one closed, or with both closed. In this way, the three substates, equally spaced in conductance, are generated. According to this view, the protochannels must be intimately associated in a complex because of the channel's bursting behavior: the simultaneous appearance and disappearance of both protochannels.This "double-barreled shotgun" model for the channel is quite eccentric, but it is well supported by several lines of evidence (4, 5): the equal spacing in conductance of the three substates, the binomially distributed probabilities of the appearance of the substates, and the "dimeric" transition probabilities among the substates. But this stochastic evidence is, ultimately, indirect. In this report, we present much stronger evidence for the dimeric structure of this channel by examining the action of 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) on the channel's substate behavior. We argue that this irreversible inhibitor can be used to trap a "half-inhibited" channel complex in which only one of the protochannels is able to function and, thus, may be observed directly.MATERIALS AND METHODS Biochemical. Noninnervated face vesicles of T. californica electroplax were prepared immediately after dissection of the tissue...

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A Voltage‐dependent Chloride Conductance Channel From Torpedo Electroplax Membrane*

Cited by 126 publications

References 17 publications

Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.

Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.

CLC-mediated anion transport in plant cells

Dimeric structure of single chloride channels from Torpedo electroplax.

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