The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein

Villegas, Raimundo; Sorais-Landáez, Françoise; Rodríguez-Grille, JoséM.; Villegas, Gloria M.

doi:10.1016/0005-2736(88)90175-7

Cited by 7 publications

(3 citation statements)

References 15 publications

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“…Nerves were dissected from lobster walking legs of the species Panulirus argus, and their plasma membranes were isolated as previously described (Villegas, Sorais-Landaez, Rodriguez-Grille, and Villegas, 1988). The isolated plasma membranes were resuspended (3--4 mg protein/ml) in sucrose buffer (0.33 M sucrose, 25 mM potassium phosphate, pH 7.4) and stored at -80°C.…”

Section: Preparation Of Sodium Channel Materialsmentioning

confidence: 99%

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers.

Castillo

Villegas

Recio-Pinto

1992

The Journal of General Physiology

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A B S T R A C T Alkaloid-modified, voltage-dependent sodium channels from lobster walking leg nerves were studied in planar neutral lipid bilayers. In symmetrical 0.5 M NaCI the single channel conductance of veratridine (VTD) (10 pS) was less than that of batrachotoxin (BTX) (16 pS) modified channels. At positive potentials, VTDbut not BTX-modified channels remained open at a flickery substate. VTD-modified channels underwent closures on the order of milliseconds (fast process), seconds (slow process), and minutes. The channel fractional open time (fo) due to the fast process, the slow process, and all channel closures (overall)Co) increased with depolarization. The fast process had a midpoint potential (Va) of -122 mV and an apparent gating charge (Za) of 2.9, and the slow process had a Va of --95 mV and a za of 1.6. The overall fo was predominantly determined by closures on the order of minutes, and had a Va of about -24 mV and a shallow voltage dependence (Za = 0.7). Augmenting the VTD concentration increased the overall fo without changing the number of detectable channels. However, the occurrence of closures on the order of minutes persisted even at super-saturating concentrations of VTD. The occurrence of these long closures was nonrandom and the level of nonrandomness was usually unaffected by the number of channels, suggesting that channel behavior was nonindependent. BTX-modified channels also underwent closures on the order of milliseconds, seconds, and minutes. Their characterization, however, was complicated by the apparent low BTX binding affinity and by an apparent high binding reversibility (channel disappearance) of BTX to these channels. VTD-but not BTX-modified channels inactivated slowly at high positive potentials (greater than +30 mV). Single channel conductance versus NaCI concentrations saturated at high NaC1 concentrations and was non-Langmuirian at low NaCI concentrations. At all NaC1 concentrations the conductance of VTD-modified channels was lower than that of BTX-modified channels. However, this difference in conductance decreased as NaCl concentrations neared zero, approaching the same limiting value. The permeability ratio of sodium over potassium obtained under mixed ionic conditions was similar for VTD (2.46)-and BTX (2.48)-modified channels, whereas that obtained under bi-ionic conditions was lower for VTD (1.83)-than for BTX Address reprint requests to Dr.

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Section: Preparation Of Sodium Channel Materialsmentioning

confidence: 99%

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers.

Castillo

Villegas

Recio-Pinto

1992

The Journal of General Physiology

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“…Knowledge about the structure and function of the Na + channel at the molecular level has advanced, thanks to the solubilization and purification of Na + channels from different sources (Agnew et al, 1978;Barchi, Cohen & Murphy, 1980;Hartshorne & Catterall, 1981;Lombet & Lazdunski, 1984;Villegas et al, 1988), to their functional reconstitution into liposomes or planar lipid bilayers (Villegas et al, 1977;Hartshorne & Caterall, 1981;Weigele & Barchi, 1982; 1984a,b; Hartshorne et al, 1985) and, more recently, to the sequencing of its DNA precursor (Noda et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

The interaction of homologous series of alkanols with sodium channels in nerve membrane vesicles

1988

Self Cite

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The potency of members of the homologous series of alkanols to block 22Na uptake through sodium channels stimulated by veratridine was studied in membrane vesicles obtained from lobster walking leg nerves. A cut-off was revealed at the level of 1-undecanol. However, secondary isomers of inactive primary homologues, such as 5-dodecanol and 5-tridecanol, were able to block ion flux. From the concentration required for an equipotent effect, it was calculated that the standard free energy for adsorption of primary alkanols is -725 cal/mol CH2. Furthermore, since the concentration required for an equipotent effect for primary isomer was found to be lower than that obtained for secondary isomers, it is concluded that the latter are less potent than the former. The similarity between this set of results and those obtained in intact frog sciatic nerve (J. Requena et al., J. Membrane Biol., 84:229-238, 1985) offers further support to the notion that the procedure employed to isolate the membrane vesicles does preserve the Na channels. However, the mechanism of alcohol inhibition of the Na channel in isolated membrane vesicles would seem to be somewhat different from that preferred in axons. While in vesicles the block needs to be thought in terms of a reduction in the number of conducting Na channel, in axons this is considered to be the less likely mode of action, mainly because under veratridine it is not possible to invoke a shift in the steady-state activation or inactivation.

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“…The sodium channel has been, so far, purified from the electroplax of the electric eel (Agnew et al, 1978;Norman et al, 1983), skeletal muscle of the rat (Barchi, 1983) and the rabbit (Kraner et al, 1985), rat brain (Hartshorne and Catterall, 1984), chick heart (Lombet and Lazdunski, 1984), and lobster nerves (Villegas et al, 1988).…”

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confidence: 99%

Solubilization of Sodium Channel from Human Brain

Rycker

Grandfils

Bettendorff

et al. 1989

Journal of Neurochemistry

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[3H]Tetrodotoxin binds to a single class of receptor sites in homogenates of human brain with a KD of 9.1 nM at 0 degree C and a maximal binding capacity of 5.9 pmol/mg of protein. This tetrodotoxin receptor has been solubilized, and several parameters influencing the efficiency of this critical step have been studied. Treatment of brain membranes with 2% (wt/vol) Nonidet P-40 solubilizes up to 38% of the tetrodotoxin receptor sites. The duration of this solubilization step must not exceed 15 min at an optimal pH of 6.8. The binding activity is most stable when exogenous phosphatidylcholine is added to the soluble receptor with a phosphatidylcholine/detergent ratio of 1:5.

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The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein

Cited by 7 publications

References 15 publications

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers.

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers.

The interaction of homologous series of alkanols with sodium channels in nerve membrane vesicles

Solubilization of Sodium Channel from Human Brain

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