Reconstitution of a KATP channel from basolateral membranes of Necturus enterocytes

1995 ◽  
Vol 269 (2) ◽  
pp. C464-C471 ◽  
Author(s):  
O. Mayorga-Wark ◽  
W. P. Dubinsky ◽  
S. G. Schultz
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
K Channel ◽  
Intestinal Epithelial ◽  
Open Time ◽  
Extracellular Compartment ◽  
Solution Bathing ◽  
Small Intestinal ◽  
K Channel Opener

We have previously reported that basolateral membrane vesicles isolated from Necturus maculosa small intestinal epithelial cells and incorporated into planar phospholipid bilayers display a highly selective "maxi"-conductance K+ channel whose open-time probability is affected by voltage. We now report that this channel is inhibited by MgATP in the solution bathing the intracellular face of the channel but not by Mg2+ or the Na+ or K+ salts of ATP; the effects of MgATP can be prevented or reversed by MgADP. The channel is also inhibited by the nonhydrolyzable ATP analogue magnesium adenosine 5'-O-(3-thiotriphosphate) and the sulfonylurea derivatives tolbutamide and glibenclamide; all of these agents are effective in the intracellular compartment but not when added to the extracellular compartment alone. Channel activity is stimulated by the "K+ channel opener," diazoxide, which also reverses the effect of glibenclamide but not of MgATP. The possible role of this channel as a mediator of the parallelism between basolateral membrane Na(+)-K+ pump activity and the macroscopic K+ conductance of that barrier is discussed.

scholarly journals Colocalization of glycolytic enzyme activity and KATP channels in basolateral membrane of Necturusenterocytes

1998 ◽  
Vol 275 (6) ◽  
pp. C1653-C1659 ◽  
Author(s):  
William P. Dubinsky ◽  
O. Mayorga-Wark ◽  
Stanley G. Schultz
Keyword(s):  
Intestinal Epithelial Cells ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Katp Channels ◽  
Glycolytic Enzyme ◽  
Phospholipid Bilayers ◽  
Intestinal Epithelial ◽  
Irreversible Inhibitor ◽  
Solution Bathing ◽  
Small Intestinal

86Rb fluxes through ATP-regulated K+(KATP) channels in membrane vesicles derived from basolateral membranes of Necturus small intestinal epithelial cells as well as the activity of single KATP channels reconstituted into planar phospholipid bilayers are inhibited by the presence of ADP plus phospho enolpyruvate in the solution bathing the inner surface of these channels. This inhibition can be prevented by pretreatment of the membranes with 2,3-butanedione, an irreversible inhibitor of pyruvate kinase (PK) and reversed by the addition of 2-deoxyglucose plus hexokinase. The results of additional studies indicate that PK activity appears to be tightly associated with this membrane fraction. These results, together with considerations of the possible ratio of Na+-K+pumps to KATP channels in the basolateral membrane, raise the possibility that “cross talk” between those channels and pumps (i.e., the “pump-leak parallelism”) may be mediated by local, functionally compartmentalized ATP-to-ADP ratios that differ from those in the bulk cytoplasm.

Effect of trypsin on a Ca(2+)-activated K+ channel reconstituted into planar phospholipid bilayers

1992 ◽  
Vol 262 (4) ◽  
pp. C971-C974 ◽  
Author(s):  
L. Salomao ◽  
G. Wark ◽  
W. P. Dubinsky ◽  
S. G. Schultz
Keyword(s):  
High Selectivity ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Phospholipid Bilayers ◽  
K Channel ◽  
Voltage Sensitivity ◽  
Ca Channel ◽  
Rat Skeletal Muscle ◽  
Cytoplasmic Side

Exposure of the cytoplasmic side of calcium-activated, high (maxi)-conductance potassium [BK(Ca)] channels in basolateral membrane vesicles from rabbit colonocytes incorporated into planar phospholipid bilayers to trypsin rapidly reduces, but does not abolish, the sensitivity of this channel to activation by calcium without affecting its conductance or high selectivity for K+ over Cl-. The results of these studies also indicate that this BK(Ca) channel does not have intrinsic voltage-gating properties but that its voltage sensitivity is related to its ability to interact with calcium. This conclusion is consistent with the model proposed by Moczydlowski and Lattore (J. Gen. Physiol. 82: 511-542, 1983) for the role of membrane voltage in modulating the interaction between calcium and the BK(Ca) channel in rat skeletal muscle.

Effects of a Shaker K+ channel peptide and trypsin on a K+ channel in Necturus enterocytes

1993 ◽  
Vol 265 (2) ◽  
pp. C541-C547 ◽  
Author(s):  
O. Mayorga-Wark ◽  
J. Costantin ◽  
W. P. Dubinsky ◽  
S. G. Schultz
Keyword(s):  
Lipid Bilayers ◽  
Basolateral Membrane ◽  
Companion Paper ◽  
K Channel ◽  
Intestinal Epithelial ◽  
K Channels ◽  
Voltage Dependent ◽  
Small Intestinal ◽  
Pore Blocker ◽  
Voltage Gate

We have previously demonstrated that a synthetic peptide composed of the first 22 amino acids from the NH2-terminus of the Shaker B K+ channel protein deactivates a voltage-dependent K+ channel present in basolateral membrane of Necturus small intestinal epithelial cells reconstituted into planar lipid bilayers (Dubinsky et al. Proc. Natl. Acad. Sci. USA 89: 1770-1774, 1992). We now demonstrate that this peptide interacts with the inner surface of the Necturus channel only when it is in the open or conducting configuration and that this interaction is hindered by tetraethylammonium ion, a well-established blocker of this and other K+ channels. We conclude that this peptide is an open-pore blocker of the Necturus K+ channel as it appears to be in the case of the Shaker B K+ channel. We further demonstrate that trypsin, which abolishes the ability of this peptide to block both the Necturus and the Shaker K+ channels and inhibits spontaneous inactivation of the Shaker K+ channel, also impairs the voltage-gate of the Necturus K+ channel. These findings, and others to be reported in a companion paper, suggest structural homologies between the "inactivation peptide" of the Shaker B K+ channel and the voltage-gate of the Necturus K+ channel.

Basolateral potassium channels in renal proximal tubule

1987 ◽  
Vol 253 (3) ◽  
pp. F476-F487 ◽  
Author(s):  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
K Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
K Channels ◽  
Open Time ◽  
Excised Patches ◽  
The Mean ◽  
Inside Out

Potassium (K+) channels in the basolateral membrane of unperfused Necturus proximal tubules were studied in both cell-attached and excised patches, after removal of the tubule basement membrane by manual dissection without collagenase. Two different K+ channels were identified on the basis of their kinetics: a short open-time K+ channel, with a mean open time less than 1 ms, and a long open-time K+ channel with a mean open time greater than 20 ms. The short open-time channel occurred more frequently than the longer channel, especially in excised patches. For inside-out excised patches with Cl- replaced by gluconate, the current-voltage relation of the short open-time K+ channel was linear over +/- 60 mV, with a K+-Na+ selectivity of 12 +/- 2 (n = 12), as calculated from the reversal potential with oppositely directed Na+ and K+ gradients. With K-Ringer in the patch pipette and Na-Ringer in the bath, the conductance of the short open-time channel was 47 +/- 2 pS (n = 15) for cell-attached patches, 26 +/- 2 pS (n = 15) for patches excised (inside out) into Na-Ringer, and 36 +/- 6 pS (n = 3) for excised patches with K-Ringer on both sides. These different conductances can be partially explained by a dependence of single-channel conductance on the K+ concentration on the interior side of the membrane. In experiments with a constant K+ gradient across excised patches, large changes in Na+ at the interior side of the membrane produced no change in single-channel conductance, arguing against a direct block of the K+ channel by Na+. Finally, the activity of the short open-time channel was voltage gated, where the mean number of open channels decreased as a linear function of basolateral membrane depolarization for potentials between -60 and 0 mV. Depolarization from -60 to -40 mV decreased the mean number of open K+ channels by 28 +/- 8% (n = 6).

Influence of vascular and luminal hexoses on rat intestinal basolateral glucose transport

1994 ◽  
Vol 72 (4) ◽  
pp. 317-326 ◽  
Author(s):  
Raymond Tsang ◽  
Ziliang Ao ◽  
Chris Cheeseman
Keyword(s):  
Glucose Transport ◽  
Plasma Glucose ◽  
Intestinal Adaptation ◽  
Basolateral Membrane ◽  
Physiological Role ◽  
Intestinal Transport ◽  
Membrane Vesicles ◽  
Luminal Perfusion

The influence of luminal and vascular hexoses in rats on glucose transport across the jejunal basolateral membrane (BLM) was measured using isolated membrane vesicles prepared from infused animals. In vivo vascular infusions of glucose produced an increase in glucose transport across BLM vesicles. Sucrose, mannose, galactose, and fructose had no significant effect. Plasma glucose concentrations were unaffected by galactose and sucrose vascular infusions, while mannose and fructose produced a modest rise, and glucose increased plasma glucose to 20 mM. Insulin release was significantly increased by vascular infusion of glucose and fructose, while mannose produced only a small sustained rise. Sucrose and galactose had no effect. Perfusion through the lumen of the rat jejunum in vivo, for up to 4 h, with glucose, fructose, sucrose, or lactate (100 or 25 mM) produced a significant increase in the maximal rate of glucose transport (up to 4- to 5-fold) across BLMs. Galactose and mannose had no effect. Luminal glucose perfusion produced a small nonsignificant increase in glucose inhibitable cytochalasin B binding to BLM vesicles, and no change was seen in the microsomal pool of binding sites. The abundance of GLUT2 in the jejunal BLM, as determined by Western blotting, was unaffected by luminal perfusion of 100 mM glucose for 4 h. Fructose almost completely inhibited the carrier-mediated uptake of glucose in control and upregulated jejunal BLM vesicles. These results are discussed in relation to the physiological role of the upregulation of GLUT2 activity by luminal and vascular hexoses.Key words: intestinal transport, basolateral membrane, glucose transport, intestinal adaptation.

Basolateral cell membrane Ca-Na exchange in single rabbit connecting tubules

1990 ◽  
Vol 258 (6) ◽  
pp. F1497-F1503 ◽  
Author(s):  
J. E. Bourdeau ◽  
K. Lau
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Base Line ◽  
Plasma Membrane Vesicles ◽  
Tetraethylammonium Chloride ◽  
Renal Tubular Cells ◽  
A Cell ◽  
Renal Tubular ◽  
Second Peak

Studies of cortical proximal nephrons and plasma membrane vesicles suggest that a Ca-Na exchanger regulates intracellular Ca2+ concentration ([Ca2+]i) in renal tubular cells. We tested this hypothesis in isolated perfused rabbit connecting segments by measuring [Ca2+]i with fura-2. Within 2 min of replacing bath NaCl with mannitol, [Ca2+]i rose from a base line of approximately 100 nM to a peak of approximately 650 nM, then declined to a plateau of approximately 500 nM for approximately 5 min before rising to a second peak of approximately 600 nM. [Ca2+]i returned toward base line after restoring bath NaCl. Substitution of choline Cl or tetraethylammonium chloride for bath NaCl reproduced the rise in [Ca2+]i, implicating the Na+ as the mediator. Selective bath (but not lumen) Ca removal or lumen Na deletion virtually abolished these effects, suggesting that bath Na deletion causes peritubular Ca influx by a process that depends on lumen Na. Lumen Na removal lowered, whereas its repletion increased, [Ca2+]i. Smaller increments in [Ca2+]i were produced by raising lumen [Na] from 0 to 35-55 mM or from 20 to 120 mM, but not from 55 to 150 mM. Clamping bath [Ca] at approximately 100 nM abolished the rise in [Ca2+]i produced by lumen Na, corroborating the role of peritubular Ca. These results suggest a Ca influx across the basolateral membrane that is driven by a cell-to-bath [Na] gradient and that can be activated by changes in lumen [Na]. We propose that this process, in part, regulates [Ca2+]i in the rabbit connecting tubule.

scholarly journals KCNJ10 (Kir4.1) is expressed in the basolateral membrane of the cortical thick ascending limb

2015 ◽  
Vol 308 (11) ◽  
pp. F1288-F1296 ◽  
Author(s):  
Chengbiao Zhang ◽  
Lijun Wang ◽  
Xiao-Tong Su ◽  
Dao-Hong Lin ◽  
Wen-Hui Wang
Keyword(s):  
Basolateral Membrane ◽  
Reversal Potential ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Initial Part ◽  
Wild Type ◽  
Open Probability ◽  
Cell Recording ◽  
Late Part

The aim of the present study is to examine the role of Kcnj10 (Kir.4.1) in contributing to the basolateral K conductance in the cortical thick ascending limb (cTAL) using Kcnj10+/+ wild-type (WT) and Kcnj10−/− knockout (KO) mice. The patch-clamp experiments detected a 40- and an 80-pS K channel in the basolateral membrane of the cTAL. Moreover, the probability of finding the 40-pS K was significantly higher in the late part of the cTAL close to the distal convoluted tubule than those in the initial part. Immunostaining showed that Kcnj10 staining was detected in the basolateral membrane of the cTAL but the expression was not uniformly distributed. The disruption of Kcnj10 completely eliminated the 40-pS K channel but not the 80-pS K channel, suggesting the role of Kcnj10 in forming the 40-pS K channel of the cTAL. Also, the disruption of Kcnj10 increased the probability of finding the 80-pS K channel in the cTAL, especially in the late part of the cTAL. Because the channel open probability of the 80-pS K channel in KO was similar to those of WT mice, the increase in the 80-pS K channel may be achieved by increasing K channel number. The whole cell recording further showed that K reversal potential measured with 5 mM K in the bath and 140 mM K in the pipette was the same in the WT and KO mice. Moreover, Western blot and immunostaining showed that the disruption of Kcnj10 did not affect the expression of Na-K-Cl cotransporter 2 (NKCC2). We conclude that Kir.4.1 is expressed in the basolateral membrane of cTAL and that the disruption of Kir.4.1 has no significant effect on the membrane potential of the cTAL and NKCC2 expression.

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