Ion transport through pores: A rate-theory analysis

Läuger, P.

doi:10.1016/0005-2736(73)90323-4

Cited by 382 publications

(190 citation statements)

References 26 publications

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“…In their study, the dependence of the single-channel current on [Nal] was ac- Na' activity (mM) (19). Our measured dependence of reconstituted Torpedo acetylcholine receptor single-channel currents upon Na+ activity was consistent with the same kinetic model (Fig.…”

Section: Methodssupporting

confidence: 81%

Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

Tank¹,

Huganir²,

Greengard³

et al. 1983

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

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Small unilamellar vesicles containing purified and reconstituted nicotinic acetylcholine receptors from Torpedo electroplax have been fused by a simple freeze-thaw procedure to form large liposomes. Giga-seal patch-recording techniques were used to form isolated patches of liposome-membrane and to measure single-channel properties of the reconstituted receptor-ion channel complex. The observed properties are quantitatively similar to those reported for vertebrate muscle nicotinic acetylcholine receptor species recorded in situ. The results demonstrate that the pentameric complex consisting of the q218ft subunits is fully functional. The methods used in these experiments should be useful in studying the effects of chemical alterations on the properties of acetylcholine receptor channels as well as other types of purified and reconstituted ion channels.The nicotinic acetylcholine receptor from Torpedo electroplax is a chemically gated ion channel consisting of a pentameric complex of the five subunits a2fy8S (for reviews, see refs. 1 and 2). It is reproducibly purified and, when reconstituted into small liposomes or planar lipid bilayers, it exhibits the basic agonistinduced cation transport properties anticipated from electrophysiological measurements in vivo (3-10). By applying recently developed methods (11, 12) for patch-recording from large liposomes containing reconstituted ion channels, we have now measured, with high resolution, single-channel properties of the purified and reconstituted Torpedo acetylcholine receptor. We report here that this pentameric complex showed an agonist-independent main conductance level, a subconductance state, saturation of the single-channel conductance at high Na+ concentration, slow inactivation and burst-kinetics at desensitizing concentrations of agonists, and open channel lifetime distributions that are not described by single exponentials. The similarity of these properties with those reported for other vertebrate acetylcholine receptor species recorded in situ provides further evidence that the purified receptor is fully functional and that the well-characterized biochemical properties associated with this isolated receptor complex may apply to a wide class of nicotinic receptor ion channels. MATERIALS AND METHODSPreparation of Liposomes Containing Reconstituted Acetylcholine Receptor. Acetylcholine receptor was isolated, purified, and reconstituted as described (4). Briefly, postsynaptic membranes rich in acetylcholine receptor were prepared from freshly dissected electric organ of Torpedo Californica. The receptor was then purified from a Na cholate extract of the postsynaptic membranes by affinity chromatography on a choline carboxymethyl affinity gel as described by Huganir and Racker (4), except that the loaded column was washed with 20 column. vol of "wash buffer" before elution. The purified and solubilized receptor was incorporated into asolectin (Associated Concentrates, Woodside, NY) small unilamellar vesicles by the detergent dialysis technique. The ...

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Section: Methodssupporting

confidence: 81%

Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

Tank¹,

Huganir²,

Greengard³

et al. 1983

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

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“…The voltage sensitivity of the block is not greater than that seen with procaine (7) or QX-222 (11). This suggests that only one of the cationic heads of DECA enters the channel, in agreement with the expectations of an elementary electrostatic mechanism (26). It is unlikely that the other head is within easy reach of the anionic binding site on the receptor, because then one would expect nearly zero-order behavior of 1 /Trf .…”

Section: Discussionsupporting

confidence: 69%

Decamethonium both opens and blocks endplate channels.

Adams¹,

Sakmann²

1978

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

148

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Miniature endplate currents, endplate current fluctuations ("membrane noise"), and voltage-jump current relaxations were studied in voltage-clamped frog muscle fibers during decamethonium action. All three types of experiments revealed two kinetic processes controlling the opening of endplate channels, one that reflects agonist action and another that reflects local anesthetic-like blocking activity. The kinetic constants for these two steps were evaluated from measurements of the fast and slow time constants as a function of decamethonium concentration. At -130 mV membrane potential and 130, the mean open time of decamethonium-activated channels is 2.8 msec. The forward and backward rate constants for channel blocking are -1.7 X 107 M l sec. and 103 sec-1. ITe voltage dependencies of the channel lifetime and of the blocking equilibrium are similar to those seen with pure agonists and local anesthetics, respectively. The transmitter at the nerve-muscle synapse of vertebrate skeletal muscle, acetylcholine (AcCho), acts by combining with postjunctional receptors and inducing the opening of discrete ion channels (1-3).A number of other quaternary ammonium compounds mimic this action of acetylcholine, although for some of them the maximum effect is quite feeble. Compounds that produce only small maximum potential or conductance changes are termed partial agonists. It has been generally supposed that these agonists are partial because they are relatively ineffective in triggering the conformational change of the receptor necessary for channel opening, either because the rate constants for this conformational change are such that the resting state is favored (4)

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“…Although sodium and potassium channel conductances range from 2 to 50 pS (Hille, 1991), general-diffusion porin monomers at similar conditions range between 5 0 0 and 1,OOO pS (Benz & Bauer, 1988). Moreover, the former channels show flux saturation (Sakmann & Trube, 1984) and have been modeled as a sequence of energy barriers connected by binding sites (Lauger, 1973;Dani, 1986). In contrast, even the simple symmetrical two-barrier-one-site model does not appear to be justified for the general diffusion porins with a linear relation between conductance and ion concentration, which can only be described by a one-barrier model.…”

Section: Discussionmentioning

confidence: 99%

X-Ray Crystallographic and Mass Spectrometric Structure Determination and Functional Characterisation of Succinylated Porin from Rhodobacter Capsulatus: Implications for Ion Selectivity and Single-Channel Conductance

Welte

Diederichs

Przybylski

et al. 1998

New Methods for the Study of Biomolecular Complexes

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The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinylporins determined by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ionization mass spectrometry identified selective succinylation of three lysinet-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal a-amino group. The structure of a tetra-succinylated porin (TS-porin) was determined to 2.4 A and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di-succinylated porin (MS-porin) and a TS-porin are localized at the inner channel surface and are solvent-accessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic entrance of the channel. The Lys-46 residue at the central constriction loop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS-and TS-porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single-channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure-selective modification of charged residues as an efficient approach in the structure-function evaluation of ion channels, and X-ray crystallography and mass spectrometry as complementary analytical tools for defining precisely the chemically modified structures.

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Ion transport through pores: A rate-theory analysis

Cited by 382 publications

References 26 publications

Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.

Decamethonium both opens and blocks endplate channels.

X-Ray Crystallographic and Mass Spectrometric Structure Determination and Functional Characterisation of Succinylated Porin from Rhodobacter Capsulatus: Implications for Ion Selectivity and Single-Channel Conductance

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