Sodium-dependent efflux of K+ and Rb+ through the activated sodium channel of neuroblastoma cells

The specific reaction rate (J) of the acetylcholine receptor-controlled ion translocation has been determined. In eel Ringer's solution (pH 7.0) at 10C, j = 3 x 107 M'1 sec-1. j is an intrinsic constant that is characteristic of the receptor and independent of other properties of a receptor-containing cell that also determine the rates of ion translocation. Membrane vesicles (prepared , which allows one to measure elementary steps in the formation of ion channels through the cell membrane.The initial rate of acetylcholine receptor-controlled movement of ions across the membrane of a particular cell determines the initial change in transmembrane potential (1, 2) and therefore also determines whether or not the electrical signal generated by a nerve cell will be propagated (3) or, in muscle cells, whether a contraction will occur. This initial rate is characterized by the rate coefficient, JA The value of JA is determined not only by the intrinsic properties of the receptor but also by certain properties of the cell, such as the number of receptor sites and the internal volume, and also the internal and external concentrations of freely mobile inorganic ions. We have, therefore, determined the specific reaction rate, J, which is characteristic of the receptor-controlled ion translocation and independent of these other properties of nerve and muscle cells. The rate of receptor-controlled ion translocation over a wide range of both carbamoylcholine and acetylcholine concentrations can be measured (12-15). The interpretation of all these measurements has led to the formulation of a minimum mechanism (Fig. 1) that relates the formation of ligand-receptor complexes to ion translocation (13,14). The integrated rate equation (Eq. 1) based on this mechanism predicts the rate of receptor-controlled flux over the concentration ranges of carbamoylcholine (13,14) and acetylcholine that we have investigated (15).JA is the observed rate coefficient associated with the initial fast influx before the onset of inactivation (desensitization) of the receptor. J. is the observed rate coefficient associated with the slow phase that occurs after the inactivation process has gone to completion, and a is the rate coefficient for the inactivation process: JA = Jm ([AL2]

mentioning

confidence: 99%

Specific reaction rate of acetylcholine receptor-controlled ion translocation: a comparison of measurements with membrane vesicles and with muscle cells.

Hess¹,

Aoshima²,

Cash³

et al. 1981

“…Monosaccharides and 2% Me2SO were added as described for the 75-cm2 flasks. The efflux assay has been described (2,5,6). Veratridine (100 ,uM) and scorpion venom (5 tug/ml of assay medium) were used to stimulate the efflux, and tetrodotoxin (1 p.M) to inhibit the flux.…”

Section: Methodsmentioning

confidence: 99%

“…Inducing agents, such as dimethyl sulfoxide (Me2SO), have provided a method for obtaining a high degree of differentiation of mouse (1) and human (2) cultured neuroblastoma cells, making it possible to measure the active Na+ channels of populations of induced cells (2)(3)(4)(5)(6). Furthermore, because these cells can be manipulated in culture, it is possible to look for agents that inhibit the development ofthe active channels by additions to the growth medium.…”

mentioning

confidence: 99%

Specific monosaccharide inhibition of active sodium channels in neuroblastoma cells.

Giovanni¹,

Kessel²,

Glick³

1981

L-Fucose and D-galactose in low concentrations (0.27 or 2.7 mM) inhibited the induction of active Na+ channels in mouse and-human neuroblastoma cells when the monosaccharides were added to the culture medium for 4 days with the inducing agent dimethyl sulfoxide. Active Na+ ionophores were determined by measurement of the toxin-stimulated efflux of 'Rb from the cells. At the same time, the amount of a radioactive glycoprotein (Mr 200,000), which was shown previously to be associated with neurite and. membrane preparations from cells with active Na+ channels, was decreased. Cell growth and viability were not affected. The nonphysiological isomer D-fucose or the addition of D-glucose in the same concentration did not inhibit differentiation. Vibriocholerae neuraminidase, added to the cells prior to the stimulation of "Rb efflux by veratridine and scorpion venom, was inhibitory. The implications of these findings, which suggest a key role for glycoproteins in at least a portion of the excitability process, are discussed.

“…The flow of each of these ions is responsive to neurotoxins and thus the effluxes of Rb+ from cells in culture (8) and Cs' from phospholipid vesicles (9) (2). Cell counts and viability determinations-were in a hemocytometer.…”

mentioning

confidence: 99%

Reconstitution in vitro of neurotoxin-responsive ion efflux by using membrane glycoproteins of neuroblastoma cells.

Giovanni¹,

Glick²

1983

Glycoproteins were purified from a clonal cell line of mouse neuroblastoma, N-18, labeled Glycoproteins have been indirectly implicated as components of the voltage-sensitive Na+ channel since neurotoxin-binding receptors were partially purified on immobilized lectins (1). However, it has not been shown directly that the components of the functional Na+ channel are glycoproteins. In other studies, the modulation of a glycoprotein, Mr 200,000, during differentiation was shown for mouse (2) and human (3) showed only a 1.5-to 2.0-fold stimulation by the neurotoxins over the passive efflux constant rate. Inhibition of the active efflux was with 1 ,tM tetrodotoxin (Calbiochem). All of the data were calculated and graphed by computer (3).The cells were harvested by washing the monolavers of 20 flasks two times with 5 ml of 0.16 M NaCl per flask and were removed by shaking gently in 5 ml of 0.16 M NaCl and then centrifuging at 280 x g at 40C. The cells then were washed five times with 0.16 M NaCl and finally pelleted. The pellet of N-18 cells that was used for extraction of the glycoproteins conAbbreviations: NP-40, Nonidet P-40; WGA, wheat germ agglutinin.