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.

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.

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.

Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels

1996 ◽  
Vol 270 (6) ◽  
pp. F1066-F1072 ◽  
Author(s):  
L. Zimniak ◽  
C. J. Winters ◽  
W. B. Reeves ◽  
T. E. Andreoli
Keyword(s):  
Lipid Bilayers ◽  
Antisense Oligonucleotide ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Western Blots ◽  
Cl Channel ◽  
Medullary Thick Ascending Limb ◽  
Cl Channels

The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.

Identification of a Basolateral Membrane Potassium Channel from Teleost Intestinal Epithelial Cells

1991 ◽  
Vol 159 (1) ◽  
pp. 45-64
Author(s):  
CHRISTOPHER A. LORETZ ◽  
CHARLES R. FOURTNER
Keyword(s):  
Membrane Potential ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Basolateral Membrane ◽  
K Channel ◽  
Ca Channel ◽  
Intestinal Epithelial ◽  
Channel Activity ◽  
Open Probability ◽  
Physiological Range

Using patch-clamp techniques, a Ca2+-dependent, voltage-gated K+ channel [K(Ca) channel] was isolated from the basolateral membrane of NaCl-absorbing intestinal epithelial cells of the goby Gillichthys mirabilis. This K(Ca) channel had a high conductance (approximately 150 pS) in the physiological range of membrane potential. Conclusive identification as a K+ channel is supported by dependence of the reversal potential for single-channel current on the K+ concentration gradient and the ability of Ba2+, Cs+ and other pharmacological agents to block the channel. The channel was highly selective for K+ over Na+ (PNa/PK=0.04). Channel activity, expressed as open probability (Po), was dependent on membrane potential with depolarization increasing Po over the physiological range in the presence of Ca2+. Channel activity was also dependent on cytoplasmic-side Ca2+. Po was reduced to near-zero levels following EGTA chelation of Ca2+ in the solution bathing the cytoplasmic face of excised membrane patches; channel activity was most sensitive to changes in Ca2+ concentration between 10nmoll−1 and 10μmoll−1. This K(Ca) channel may be one of several avenues for K+ exit across the basolateral cell membrane and, as such, may play roles in both transepithelial salt transport and maintenance of intracellular ionic composition.

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.

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