Current-voltage relations of sodium-coupled sugar transport across the apical membrane ofNecturus small intestine

Lapointe, Jean‐Yves; Hudson, Randall L.; Schultz, Stanley G.

doi:10.1007/bf01871175

Cited by 26 publications

(16 citation statements)

References 44 publications

(50 reference statements)

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“…An explanation for the initial rapid response, supported by past and present findings, is that the rapid depolarization of Emc is triggered by the activation ofrheogenic and conductive Na'-coupled sugar and/or amino acid entry mechanisms in the apical membrane (12,24). This depolarization would reduce the activity of the inward-rectifying K+ channels in the basolateral membrane which, in turn, would potentiate the depolarization of both limiting membranes.…”

Section: Methodssupporting

confidence: 78%

Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.

Costantin

Alcalen

Otero

et al. 1989

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

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Basolateral membrane vesicles from Necturus enterocytes, highly (>20-fold) enriched in Na+,K+-ATPase, were reconstituted into planar lipid bilayers. The principal channel activity observed is selective for K+ over Na+ and Cl1. This K+ channel is blocked by Ba2+ and Leiurus quinquestiiatus venom but is not affected by Ca2+ over the range of 10-3 to <10-7 M and is not inhibited by charybdotoxin. L. quinquestriatus venom also markedly reduces the conductance of the basolateral membrane of intact villus cells ofNecturus smallintestine. The open-time probability (P.) of the channel displays a voltage-dependence characteristic of an "inward rectifier"; i.e., the channel inactivates when the basolateral membrane is depolarized and PO increases with increasing hyperpolarization of that barrier. Assuming that similar properties prevail under physiological conditions, this characteristic could provide, in part, an explanation for the parallelism between Na+-pump and K+-leak activities of the basolateral membrane observed in this epithelium. Thus, an increase in rheogenic Na'-pump activity at the basolateral membrane would hyperpolarize that barrier and, in turn, increase the open time of this K+ channel.The results of electrophysiological studies on a variety of Na'-absorbing epithelial cells have disclosed parallelisms among the rates of Na' entry across their apical membranes, the Na'-pump activities at their basolateral membranes, and the K+ conductances of their basolateral membranes. Although the physiological utility of these "pump-leak" parallelisms is clear, their underlying mechanisms have not been resolved (1, 2).Two methods have been developed that permit the investigation of the properties and regulation of single ionic channels at the molecular level-i.e., the patch-clamp technique and techniques for reconstituting ionic channels into planar phospholipid bilayers. The application of the patchclamp technique to the study of single-channel activities in the basolateral membranes of Na'-absorbing epithelial cells is, however, restricted by the fact that in many instances those membranes are bounded by subepithelial layers of connective tissue, smooth muscle, etc., which preclude the formation of gigaohm seals.In this paper we describe a method for isolating a highly enriched preparation of basolateral membranes from Necturus enterocytes, the reconstitution of these vesicles into planar phospholipid bilayers, and the general properties of a K+ channel present in those membranes. METHODSIsolation of Basolateral Membranes. Male Necturus maculosa were anesthetized by immersion in a 0.1% tricaine solution. The small intestine was removed and immersed in an ice-cold amphibian Ringer's solution, and the mucosal cells were scraped off with a glass slide and transferred to an isolation medium consisting of 180 mM sucrose and 2 mM Tris adjusted to pH 7.4 with Hepes (Tris/Hepes). All of the subsequent steps in the isolation procedure were carried out at 40C.The membrane fractionation procedure employed is a modific...

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Section: Methodssupporting

confidence: 78%

Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.

Costantin

Alcalen

Otero

et al. 1989

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

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“…These responses are followed by a slower, partial repolarization of mc that is paralleled by an increase in (rm/rs) to levels that exceed those observed in the absence of the sugar or amino acid (1). The initial responses can be attributed to the activation of rheogenic and conductive carrier-mediated pathways for the coupled entry of Na+ and sugars or amino acids into the cell across the apical membrane (1,2). The secondary responses appear to be, at least in part, due to an increase in the conductance of the basolateral membranes to K+, (gk), which is blocked by the presence of Ba2+ in the serosal solution and by exposure of the tissue to metabolic inhibitors (1,3,4).…”

mentioning

confidence: 98%

Sodium-coupled glycine uptake by Ehrlich ascites tumor cells results in an increase in cell volume and plasma membrane channel activities.

Hudson

Schultz

1988

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

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The addition of 10 mM glycine to a physiological saline bathing Ehrlich ascites tumor cells is followed by a slow increase in cell volume that plateaus between 15 and 30 min at a level -17% greater than the control volume; this increase is not observed when glycine is added to cells suspended in a Na+-free saline. The results of studies using the patch-clamp technique in the cell-attached mode indicate that, 0.5-3 min after the addition of glycine to the bathing solution, there is a marked increase in the activity of single channels, which in almost all instances were previously present and operant in the plasma membrane. Successfully excised patches ofmembrane that contained a channel stimulated by glycine fell into two categories. Some became inactive within 15 sec in spite of the fact that the Gil seal remained intact. Others persisted for the lifetime of the seal. All of the persistent channels had an 11-fold selectivity for ClP over K+ and a conductance of 23 pS when bathed by symmetrical 150 mM KCI solutions. Although the ionic specificities of the other channels have not been identified, there is reason to suspect that they might be K+ channels whose activities are dependent on factors lost when the patch is excised. Swelling induced by exposing these cells to a 50% hypotonic perfusate stimulated the activities of Clchannels whose properties closely resemble those stimulated by the addition of glycine to the perfusate, strongly suggesting that the glycine-induced stimulation of Cl-channel activity is part of a volume-regulatory response to cell swelling. If the increase in channel activity induced by the addition of glycine to the perfusate is indeed a response to cell swelling, then this volume-regulatory response must be extremely sensitive inasmuch as it appears to be "triggered" by an average increase in cell volume that does not exceed 5%.The results of previous studies reported by this laboratory indicate that the addition of sugars or amino acids to the solution bathing the mucosal surface of Necturus small intestine results in an initial, rapid depolarization of the electrical potential difference across the apical membrane (f/c) and a decrease in the ratio of the resistance of the apical membrane (rm) to that of the basolateral membrane (rs) i.e., (rI/rs). These responses are followed by a slower, partial repolarization of mc that is paralleled by an increase in (rm/rs) to levels that exceed those observed in the absence of the sugar or amino acid (1). The initial responses can be attributed to the activation of rheogenic and conductive carrier-mediated pathways for the coupled entry of Na+ and sugars or amino acids into the cell across the apical membrane (1, 2). The secondary responses appear to be, at least in part, due to an increase in the conductance of the basolateral membranes to K+, (gk), which is blocked by the presence of Ba2+ in the serosal solution and by exposure of the tissue to metabolic inhibitors (1, 3, 4).In addition, circumstantial evidence has been reported suggesting...

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“…5B demonstrated a voltage dependence of the Na GABA co-transport. u is an indicator of this co-transport activity (eqn (1) carrier-mediated mechanisms such as the Na-Ca counter-transporter (Deitmer & Ellis, 1978) and the sodium galactose co-transporter (Lapointe, Hundson & Schultz, 1986) are known to operate in either direction. It is also of interest to cite the [Na']0-dependent release of GABA from retinal horizontal cells (Yazulla, 1985).…”

Section: And [Gaba]bulkmentioning

confidence: 99%

Sodium‐dependent suppression of gamma‐aminobutyric‐acid‐gated chloride currents in internally perfused frog sensory neurones.

Akaike

Maruyama

Sikdar

et al. 1987

The Journal of Physiology

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SUMMARY1. The effects of the Na+ electrochemical potential gradient on y-aminobutyric acid (GABA)-induced C1-currents (ICI) in frog sensory neurones were studied, using a suction pipette technique with which internal perfusion can be accomplished under current-and voltage-clamp conditions.2. Under current clamp, the depolarizing response to GABA decreased in the presence of external Na+. A similar external Na+-dependent reduction in the GABAinduced inward ICi was observed under voltage clamp. The reversal potential of GABA-induced Ich (EGABA) was nearly equal to the Cl-equilibrium potential (Ec1), irrespective of the presence or absence of external Na+.3. Varying the Na+ influx by changing the holding membrane potential (VH) altered the GABA response: the GABA-induced Ic, decreased progressively as VH became more negative. ICi was mediated chiefly by the uptake of GABA subserved by a Na-GABA cotransport mechanism. 7. GABA dose-response measurements were made with and without external Na+.The [Na+]0-induced suppression was more pronounced in relative amount at lower concentrations and in absolute amount at intermediate concentrations. Analysis of these data indicates, however, that the Na+-coupled GABA influx kept increasing at GABA concentrations high enough to nearly saturate GABA-induced Icl, and the same saturating level was observed as in the Na+-free case. This indicates that the electrogenic co-transport current was much smaller so that our measure-

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Current-voltage relations of sodium-coupled sugar transport across the apical membrane ofNecturus small intestine

Cited by 26 publications

References 44 publications

Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.

Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.

Sodium-coupled glycine uptake by Ehrlich ascites tumor cells results in an increase in cell volume and plasma membrane channel activities.

Sodium‐dependent suppression of gamma‐aminobutyric‐acid‐gated chloride currents in internally perfused frog sensory neurones.

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