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.

scholarly journals Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli.

1992 ◽  
Vol 99 (4) ◽  
pp. 591-613 ◽  
Author(s):  
T A Cummings ◽  
S C Kinnamon
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Taste Receptor ◽  
K Channel ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
K Channels ◽  
Voltage Dependent ◽  
Whole Cell Recordings ◽  
Inside Out

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.

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 Mechanism of Maxi-K Channel Activation by Dehydrosoyasaponin-I

1998 ◽  
Vol 112 (4) ◽  
pp. 485-501 ◽  
Author(s):  
Kathleen M. Giangiacomo ◽  
Augustus Kamassah ◽  
Guy Harris ◽  
Owen B. McManus
Keyword(s):  
Lipid Bilayers ◽  
Protein Structures ◽  
Channel Gating ◽  
High Order ◽  
K Channel ◽  
Single Channels ◽  
Open Probability ◽  
Channel Activation ◽  
K Channels ◽  
Voltage Dependent

Dehydrosoyasaponin-I (DHS-I) is a potent activator of high-conductance, calcium-activated potassium (maxi-K) channels. Interaction of DHS-I with maxi-K channels from bovine aortic smooth muscle was studied after incorporating single channels into planar lipid bilayers. Nanomolar amounts of intracellular DHS-I caused the appearance of discrete episodes of high channel open probability interrupted by periods of apparently normal activity. Statistical analysis of these periods revealed two clearly separable gating modes that likely reflect binding and unbinding of DHS-I. Kinetic analysis of durations of DHS-I-modified modes suggested DHS-I activates maxi-K channels through a high-order reaction. Average durations of DHS-I-modified modes increased with DHS-I concentration, and distributions of these mode durations contained two or more exponential components. In addition, dose-dependent increases in channel open probability from low initial values were high order with average Hill slopes of 2.4–2.9 under different conditions, suggesting at least three to four DHS-I molecules bind to maximally activate the channel. Changes in membrane potential over a 60-mV range appeared to have little effect on DHS-I binding. DHS-I modified calcium- and voltage-dependent channel gating. 100 nM DHS-I caused a threefold decrease in concentration of calcium required to half maximally open channels. DHS-I shifted the midpoint voltage for channel opening to more hyperpolarized potentials with a maximum shift of −105 mV. 100 nM DHS-I had a larger effect on voltage-dependent compared with calcium-dependent channel gating, suggesting DHS-I may differentiate these gating mechanisms. A model specifying four identical, noninteracting binding sites, where DHS-I binds to open conformations with 10–20-fold higher affinity than to closed conformations, explained changes in voltage-dependent gating and DHS-I-induced modes. This model of channel activation by DHS-I may provide a framework for understanding protein structures underlying maxi-K channel gating, and may provide a basis for understanding ligand activation of other ion channels.

Ba2+, TEA+, and quinine effects on apical membrane K+ conductance and maxi K+ channels in gallbladder epithelium

1990 ◽  
Vol 259 (1) ◽  
pp. C56-C68 ◽  
Author(s):  
Y. Segal ◽  
L. Reuss
Keyword(s):  
Apical Membrane ◽  
Membrane Conductance ◽  
Membrane Resistance ◽  
K Channel ◽  
Dependent Manner ◽  
Gallbladder Epithelium ◽  
Concentration Dependent Manner ◽  
K Channels ◽  
Voltage Dependent ◽  
Channel Currents

The apical membrane of Necturus gallbladder epithelium contains a voltage-activated K+ conductance [Ga(V)]. Large-conductance (maxi) K+ channels underlie Ga(V) and account for 17% of the membrane conductance (Ga) under control conditions. We examined the Ba2+, tetraethylammonium (TEA+), and quinine sensitivities of Ga and single maxi K+ channels. Mucosal Ba2+ addition decreased resting Ga in a concentration-dependent manner (65% block at 5 mM) and decreased Ga(V) in a concentration- and voltage-dependent manner. Mucosal TEA+ addition also decreased control Ga (60% reduction at 5 mM). TEA+ block of Ga(V) was more potent and less voltage dependent that Ba2+ block. Maxi K+ channels were blocked by external Ba2+ at millimolar levels and by external TEA+ at submillimolar levels. At 0.3 mM, quinine (mucosal addition) hyperpolarized the cell membranes by 6 mV and reduced the fractional apical membrane resistance by 50%, suggesting activation of an apical membrane K+ conductance. At 1 mM, quinine both activated and blocked K(+)-conductive pathways. Quinine blocked maxi K+ channel currents at submillimolar concentrations. We conclude that 1) Ba2+ and TEA+ block maxi K+ channels and other K+ channels underlying resting Ga; 2) parallels between the Ba2+ and TEA+ sensitivities of Ga(V) and maxi K+ channels support a role for these channels in Ga(V); and 3) quinine has multiple effects on K(+)-conductive pathways in gallbladder epithelium, which are only partially explained by block of apical membrane maxi K+ channels.

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).

Arachidonic acid inhibits K channels in basolateral membrane of the thick ascending limb

2002 ◽  
Vol 283 (3) ◽  
pp. F407-F414 ◽  
Author(s):  
Rui-Min Gu ◽  
Wen-Hui Wang
Keyword(s):  
Arachidonic Acid ◽  
Basolateral Membrane ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Cytochrome P 450

We have used the patch-clamp technique to study the effect of arachidonic acid (AA) on the basolateral K channels in the medullary thick ascending limb (mTAL) of rat kidney. An inwardly rectifying 50-pS K channel was identified in cell-attached and inside-out patches in the basolateral membrane of the mTAL. The channel open probability ( P o) was 0.51 at the spontaneous cell membrane potential and decreased to 0.25 by 30 mV hyperpolarization. The addition of 5 μM AA decreased channel activity, identified as NP o, from 0.58 to 0.08 in cell-attached patches. The effect of AA on the 50-pS K channel was specific because 10 μM cis-11,14,17-eicosatrienoic acid had no significant effect on channel activity. To determine whether the effect of AA was mediated by AA per se or by its metabolites, we examined the effect of AA on channel activity in the presence of indomethacin, an inhibitor of cyclooxygenase, or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), an inhibitor of cytochrome P-450 monooxygenase. Inhibition of cyclooxygenase increased channel activity from 0.54 to 0.9. However, indomethacin did not abolish the inhibitory effect of AA on the 50-pS K channel. In contrast, inhibition of cytochrome P-450 metabolism not only increased channel activity from 0.49 to 0.83 but also completely abolished the effect of AA. Moreover, addition of DDMS can reverse the inhibitory effect of AA on channel activity. The notion that the effect of AA was mediated by cytochrome P-450-dependent metabolites of AA is also supported by the observation that addition of 100 nM of 20-hydroxyeicosatetraenoic acid, a main metabolite of AA in the mTAL, can mimic the effect of AA. We conclude that AA inhibits the 50-pS K channel in the basolateral membrane of the mTAL and that the effect of AA is mainly mediated by cytochrome P-450-dependent metabolites of AA.

scholarly journals Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

1992 ◽  
Vol 100 (3) ◽  
pp. 401-426 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Glomus Cells ◽  
Control Value ◽  
Voltage Dependent ◽  
K Current ◽  
Chemoreceptor Cells

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

Internal and external TEA block in single cloned K+ channels

1991 ◽  
Vol 261 (4) ◽  
pp. C583-C590 ◽  
Author(s):  
G. E. Kirsch ◽  
M. Taglialatela ◽  
A. M. Brown
Keyword(s):  
Single Channel ◽  
Channel Structure ◽  
K Channel ◽  
Cdna Clones ◽  
K Channels ◽  
Open Time ◽  
Voltage Dependent ◽  
Internal Site ◽  
Channel Open Time ◽  
Single Channel Currents

Tetraethylammonium (TEA) has been used recently to probe natural and mutational variants of voltage-dependent K+ channels encoded by cDNA clones. Its usefulness as a probe of channel structure prompted us to examine the molecular mechanism by which TEA blocks single-channel currents in Xenopus oocytes expressing the rat brain K+ channel, RCK2. TEA at the intracellular surface of membrane patches decreased channel open time and increased the duration of closed intervals. Tetrapentylammonium had similar but more potent effects. Extracellular application of TEA caused an apparent reduction of single-channel amplitude. Block was slower at the high-affinity internal site than at the low-affinity external site. Internal TEA selectively blocks open K+ channels, and the voltage dependence of the block indicates that the binding site lies within the membrane electric field at a point 25% of the distance from the cytoplasmic margin. External TEA also interacts with the open channel but is less sensitive to membrane potential. The results indicate that the internal and external TEA binding sites define the inner and outer margins of the aqueous pore.

Adenosine stimulates the basolateral 50 pS K channels in the thick ascending limb of the rat kidney

2007 ◽  
Vol 293 (1) ◽  
pp. F299-F305 ◽  
Author(s):  
Ruimin Gu ◽  
Jing Wang ◽  
Yunhong Zhang ◽  
Wennan Li ◽  
Ying Xu ◽  
...  
Keyword(s):  
Adenosine Receptor ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Adenosine Receptor Agonist

We used the patch-clamp technique to examine the effect of adenosine on the basolateral K channels in the thick ascending limb (TAL) of the rat kidney. A 50-pS inwardly rectifying K channel was detected in the basolateral membrane, and the channel activity was decreased by hyperpolarization. Application of adenosine (10 μM) increased the activity of basolateral 50 pS K channels, defined by NPo, from 0.21 to 0.41. The effect of adenosine on the 50 pS K channels was mimicked by cyclohexyladenosine (CHA), which increased channel activity by a dose-dependent manner. However, inhibition of the A1 adenosine receptor with 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) failed to block the effect of CHA. In contrast, application of 8-(3-chlorostyryl) caffeine (CSC), an A2 adenosine antagonist, abolished the stimulatory effect of CHA. The possibility that the effect of adenosine and adenosine analog on the basolateral 50 pS K channel was the result of activation of the A2 adenosine receptor was also suggested by the observation that application of CGS-21680, a selected A2A adenosine receptor agonist, increased the channel activity. Also, inhibition of PKA with N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide-2HC1 abolished the stimulatory effect of CHA on the basolateral 50 pS K channel. Moreover, addition of the membrane-permeable cAMP analog increases the activity of 50 pS K channels. We conclude that adenosine activates the 50 pS K channel in the basolateral membrane of the TAL and the stimulatory effect is mainly mediated by a PKA-dependent pathway via the A2 adenosine receptor in the TAL.

scholarly journals Mechanism of charybdotoxin block of the high-conductance, Ca2+-activated K+ channel.

1988 ◽  
Vol 91 (3) ◽  
pp. 335-349 ◽  
Author(s):  
R MacKinnon ◽  
C Miller
Keyword(s):  
Lipid Bilayers ◽  
External Solution ◽  
Dissociation Rate ◽  
K Channel ◽  
Internal Solution ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
K Concentration ◽  
A Site ◽  
Low K

The mechanism of charybdotoxin (CTX) block of single Ca2+-activated K+ channels from rat muscle was studied in planar lipid bilayers. CTX blocks the channel from the external solution, and K+ in the internal solution specifically relieves toxin block. The effect of K+ is due solely to an enhancement of the CTX dissociation rate. As internal K+ is raised, the CTX dissociation rate increases in a rectangular hyperbolic fashion from a minimum value at low K+ of 0.01 s-1 to a maximum value of approximately 0.2 s-1. As the membrane is depolarized, internal K+ more effectively accelerates CTX dissociation. As the membrane is hyperpolarized, the toxin dissociation rate approaches 0.01 s-1, regardless of the K+ concentration. When internal K+ is replaced by Na+, CTX dissociation is no longer voltage dependent. The permeant ion Rb also accelerates toxin dissociation from the internal solution, while the impermeant ions Li, Na, Cs, and arginine do not. These results argue that K ions can enter the CTX-blocked channel from the internal solution to reach a site located nearly all the way through the conduction pathway; when K+ occupies this site, CTX is destabilized on its blocking site by approximately 1.8 kcal/mol. The most natural way to accommodate these conclusions is to assume that CTX physically plugs the channel's externally facing mouth.

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