Primary structure and functional properties of an epithelial K channel

1994 ◽  
Vol 266 (3) ◽  
pp. C809-C824 ◽  
Author(s):  
H. Zhou ◽  
S. S. Tate ◽  
L. G. Palmer
Keyword(s):  
Functional Properties ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Rat Kidney ◽  
Reversal Potential ◽  
Ammonium Ion ◽  
Expression Cloning ◽  
K Channel ◽  
Dependent Manner ◽  
Open Probability

Expression cloning in Xenopus oocytes was used to identify a clone for a renal K channel. The clone, named ROMK2, was obtained from a cDNA library constructed in the plasmid vector pSPORT using size-selected poly(A)+ RNA from whole rat kidney. ROMK2 consists of 1,837 nucleotides, with an open reading frame of 1,116 bases predicted to code for a 372-amino acid peptide. The clone appears to be a splice variant of a recently reported K channel (ROMK1) from rat renal outer medulla (Ho, K.H., C.G. Nichols, W.J. Lederer, J. Lytton, P.M. Vassilev, M.V. Kanazirska, and S.C. Hebert. Nature Lond. 362: 31-37, 1993). Northern blot analysis indicates that ROMK2 is expressed in renal cortex, medulla, and papilla. Expression in other tissues appears to be much lower. The functional properties of the channel as measured in Xenopus oocytes indicate its close relationship to ROMK1 and more distant relationship to the inward rectifier K channel (IRK1) (Kubo, Y, T.J. Baldwin, Y. N. Jan, and L. Y. Jan. Nature Lond. 362: 127-133, 1993). The inward conductance of the channel is a saturable function of external K, with a half-maximal conductance at <5 mM. The selectivity sequence for ion permeability based on reversal potential measurements was K > Rb > NH4 > Na, Li. The conductance to Rb was only one-half that to K. Extracellular Ba2+ and Cs+ blocked the channel in a voltage-dependent manner. The high sensitivity of Cs+ block to voltage is consistent with the channel's operating as a multi-ion pore. The channel was blocked by high concentrations (100 microM) of glibenclamide. It did not appear to be blocked by extracellular Na+ or tetraethyl-ammonium ion. Patch-clamp measurements indicated a single-channel conductance of 30 pS in the presence of 110 mM K and high open probability that was weakly dependent on voltage. This channel may be involved in maintaining the membrane potential of renal cells and/or mediating renal K secretion.

Ammonium ion enhances the calcium-dependent gating of a mammalian large conductance, calcium-sensitive K+ channel

2001 ◽  
Vol 79 (11) ◽  
pp. 919-923 ◽  
Author(s):  
Andrew P Braun
Keyword(s):  
Single Channel ◽  
Ammonium Ion ◽  
K Channel ◽  
Free Calcium ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Hek 293 ◽  
Calcium Dependent ◽  
Single Channel Currents

We observed that the current amplitude and activation of expressed, mouse brain large conductance, calcium-sensitive K+ channels (BKCa channels) may be reversibly enhanced following addition of low concentrations of the weakly permeant cation NH4+ to the cytoplasmic face of the channel in excised, inside-out membrane patches from HEK 293 cells. Conductance-voltage relations were left-shifted along the voltage axis by addition of NH4Cl in a concentration-dependent manner, with an EC50 of 18.5 mM. Furthermore, this effect was observed in the presence of cytosolic free calcium (~1 µM), but was absent in a cytosolic bath solution containing nominally zero free calcium (e.g., 5 mM EGTA only), a condition under which these channels undergo largely voltage-dependent gating. Recordings of single BKCa channel events indicated that NH4+ increased the channel open probability of single channel activity ~3-fold, but did not alter the amplitude of single channel currents. These findings suggest that the calcium-sensitive gating of mammalian BKCa channels may be modified by other ions present in cytosolic solution.Key words: potassium channel, calcium, modulation, electrophysiology.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

scholarly journals Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

2009 ◽  
Vol 133 (5) ◽  
pp. 525-546 ◽  
Author(s):  
Nathaniel T. Blair ◽  
J. Stefan Kaczmarek ◽  
David E. Clapham
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Fold Increase ◽  
Receptor Activation ◽  
Brain Regions ◽  
Repetitive Firing ◽  
Dependent Manner ◽  
Open Probability ◽  
Current Voltage ◽  
Voltage Dependent

TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.

Evidence for KCNQ1 K+ channel expression in rat zymogen granule membranes and involvement in cholecystokinin-induced pancreatic acinar secretion

2008 ◽  
Vol 294 (4) ◽  
pp. C879-C892 ◽  
Author(s):  
Wing-Kee Lee ◽  
Blazej Torchalski ◽  
Eleni Roussa ◽  
Frank Thévenod
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Fluid Secretion ◽  
Pancreatic Tissue ◽  
Bk Channel ◽  
K Channel ◽  
Amylase Secretion ◽  
Zymogen Granule ◽  
Open Probability ◽  
Pancreatic Acini

Secretion of enzymes and fluid induced by Ca2+ in pancreatic acini is not completely understood and may involve activation of ion conductive pathways in zymogen granule (ZG) membranes. We hypothesized that a chromanol 293B-sensitive K+ conductance carried by a KCNQ1 protein is expressed in ZG membranes (ZGM). In suspensions of rat pancreatic ZG, ion flux was determined by ionophore-induced osmotic lysis of ZG suspended in isotonic salts. The KCNQ1 blocker 293B selectively blocked K+ permeability (IC50 of ∼10 μM). After incorporation of ZGM into planar bilayer membranes, cation channels were detected in 645/150 mM potassium gluconate cis/trans solutions. Channels had linear current-voltage relationships, a reversal potential ( Erev) of −20.9 ± 0.9 mV, and a single-channel K+ conductance ( gK) of 265.8 ± 44.0 pS ( n = 39). Replacement of cis 500 mM K+ by 500 mM Na+ shifted Erev to −2.4 ± 3.6 mV ( n = 3), indicating K+ selectivity. Single-channel analysis identified several K+ channel groups with distinct channel behaviors. K+ channels with a gK of 651.8 ± 88.0 pS, Erev of −22.9 ± 2.2 mV, and open probability ( Popen) of 0.43 ± 0.06 at 0 mV ( n = 6) and channels with a gK of 155.0 ± 11.4 pS, Erev of −18.3 ± 1.8 mV, and Popen of 0.80 ± 0.03 at 0 mV ( n = 3) were inhibited by 100 μM 293B or by the more selective inhibitor HMR-1556 but not by the maxi-Ca2+-activated K+ channel (BK channel) inhibitor charybdotoxin (5 nM). KCNQ1 protein was demonstrated by immunoperoxidase labeling of pancreatic tissue, immunogold labeling of ZG, and immunoblotting of ZGM. 293B also inhibited cholecystokinin-induced amylase secretion of permeabilized acini (IC50 of ∼10 μM). Thus KCNQ1 may account for ZG K+ conductance and contribute to pancreatic hormone-stimulated enzyme and fluid secretion.

TOK1 Is a Volatile Anesthetic Stimulated K+Channel 

1998 ◽  
Vol 88 (4) ◽  
pp. 1076-1084 ◽  
Author(s):  
Andrew T. Gray ◽  
Bruce D. Winegar ◽  
Dmitri J. Leonoudakis ◽  
John R. Forsayeth ◽  
Spencer C. Yost
Keyword(s):  
Xenopus Oocytes ◽  
Single Channel ◽  
Rank Order ◽  
Volatile Anesthetic ◽  
K Channel ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Anesthetic Agents ◽  
Electrode Voltage ◽  
Pore Domain

Background Volatile anesthetic agents can activate the S channel, a baseline potassium (K+) channel, of the marine mollusk Aplysia. To investigate whether cloned ion channels with electrophysiologic properties similar to the S channel (potassium selectivity, outward rectification, and activation independent of voltage) also are modulated by volatile anesthetic agents, the authors expressed the cloned yeast ion channel TOK1 (tandem pore domain, outwardly rectifying K+ channel) in Xenopus oocytes and studied its sensitivity to volatile agents. Methods Standard two-electrode voltage and patch clamp recording methods were used to study TOK1 channels expressed in Xenopus oocytes. Results Studies with two-electrode voltage clamp at room temperature showed that halothane, isoflurane, and desflurane increased TOK1 outward currents by 48-65% in barium Frog Ringer's perfusate. The concentrations at which 50% potentiation occurred (EC50 values) were in the range of 768-814 microM (0.016-0.044 atm) and had a rank order of potency in atm in which halothane &gt; isoflurane &gt; desflurane. The potentiation of TOK1 by volatile anesthetic agents was rapid and reversible (onset and offset, 1-20 s). In contrast, the nonanesthetic 1,2-dichlorohexafluorocyclobutane did not potentiate TOK1 currents in concentrations up to five times the MAC value predicted by the Meyer-Overton hypothesis based on oil/gas partition coefficients. Single TOK1 channel currents were recorded from excised outside-out patches. The single channel open probability increased as much as twofold in the presence of isoflurane and rapidly returned to the baseline values on washout. Volatile anesthetic agents did not alter the TOK1 single channel current-voltage (I-V) relationship, however, suggesting that the site of action does not affect the permeation pathway of the channel. Conclusion TOK1 is a potassium channel that is stimulated by volatile anesthetic agents. The concentrations over which potentiation occurred (EC50 values) were higher than those commonly used in clinical practice (approximately twice MAC).

scholarly journals Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block.

1991 ◽  
Vol 98 (1) ◽  
pp. 163-181 ◽  
Author(s):  
W B Ferguson
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Competitive Inhibition ◽  
Reversal Potential ◽  
K Channel ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Patch Clamp Technique ◽  
Channel Current ◽  
Single Channel Current

The patch-clamp technique was used to investigate the effect of intracellular Mg2+ (Mgi2+) on the conductance of the large-conductance, Ca(2+)-activated K+ channel in cultured rat skeletal muscle. Measurements of single-channel current amplitudes indicated that Mgi2+ decreased the K+ currents in a concentration-dependent manner. Increasing Mgi2+ from 0 to 5, 10, 20, and 50 mM decreased channel currents by 34%, 44%, 56%, and 73%, respectively, at +50 mV. The magnitude of the Mgi2+ block increased with depolarization. For membrane potentials of -50, +50, and +90 mV, 20 mM Mgi2+ reduced the currents 22%, 56%, and 70%, respectively. Mgi2+ did not change the reversal potential, indicating that Mg2+ does not permeate the channel. The magnitude of the Mgi2+ block decreased as the concentration of K+ was increased. At a membrane potential of +50 mv, 20 mM Mgi2+ reduced the currents 71%, 56%, and 25% for Ki+ of 75, 150, and 500 mM. These effects of Mgi2+, voltage, and K+ were totally reversible. Although the Woodhull blocking model could approximate the voltage and concentration effects of the Mgi2+ block (Kd approximately 30 mM with 150 mM symmetrical K+; electrical distance approximately 0.22 from the inner surface), the Woodhull model could not account for the effects of K+. Double reciprocal plots of 1/single channel current vs. 1/[K+] in the presence and absence of Mgi2+, indicated that the Mgi2+ block is consistent with apparent competitive inhibition between Mgi2+ and Ki+. Cai2+, Nii2+, and Sri2+ were found to have concentration- and voltage-dependent blocking effects similar, but not identical, to those of Mgi2+. These observations suggest the blocking by Mgi2+ of the large-conductance, Ca(2+)-activated K+ channel is mainly nonspecific, competitive with K+, and at least partially electrostatic in nature.

Is the secretory K channel in the rat CCT ROMK?

1997 ◽  
Vol 273 (3) ◽  
pp. F404-F410 ◽  
Author(s):  
L. G. Palmer ◽  
H. Choe ◽  
G. Frindt
Keyword(s):  
Xenopus Oocytes ◽  
Single Channel ◽  
Reversal Potential ◽  
K Channel ◽  
Sk Channels ◽  
Closed State ◽  
Open Time ◽  
Collecting Tubule ◽  
Kinetics Of ◽  
K 41

The biophysical properties of low-conductance secretory K (SK) channels in the apical membrane of the rat cortical collecting tubule were examined to compare these properties with those of the cloned renal K channels of the ROMK family expressed in oocytes. At room temperature, with the tubule superfused with 140 mM K and 110 mM cation in the pipette, the inward single-channel conductance of the SK channels was 36 +/- 1 pS for K, 41 +/- 2 pS for NH4, and 22 +/- 3 pS for Tl. The reversal potential was nearly the same for K and Tl in the pipette but was shifted by -60 mV for NH4. The kinetics of the channel when K was the permeant ion could be described by a single open state (mean open time, 24 ms) and two closed states (mean closed times, 1.6 and 65 ms). The kinetics of SK changed when Tl was the permeant ion (mean open times of 6.6 ms and no long closed state) and when NH4 was the permeant ion (mean open time of 3.0 ms and a more prevalent long closed state). Thus the gating kinetics of the channel depend strongly on the nature of the conducted ion. The properties of SK channels were quite similar to those of ROMK2 expressed in Xenopus oocytes and measured under similar conditions, suggesting that these channels are identical.

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel

1998 ◽  
Vol 274 (1) ◽  
pp. C149-C160 ◽  
Author(s):  
Daniel C. Devor ◽  
Raymond A. Frizzell
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Unsaturated Fatty Acid ◽  
Single Channel ◽  
Cell Swelling ◽  
Partial Substitution ◽  
K Channel ◽  
Dependent Manner ◽  
T84 Cells ◽  
Open Probability

We used single-channel recording techniques to identify and characterize a large-conductance, Ca2+-independent K+ channel in the colonic secretory cell line T84. In symmetric potassium gluconate, this channel had a linear current-voltage relationship with a single-channel conductance of 161 pS. Channel open probability ( P o) was increased at depolarizing potentials. Partial substitution of bath K+ with Na+ indicated a permeability ratio of K+ to Na+ of 25:1. Channel P o was reduced by extracellular Ba2+. Event-duration analysis suggested a linear kinetic model for channel gating having a single open state and three closed states: C3←→C2←→C1←→O. Arachidonic acid (AA) increased the P o of the channel, with an apparent stimulatory constant ( K s) of 1.39 μM. Neither channel open time (O) nor the fast closed time (C1) was affected by AA. In contrast, AA dramatically reduced mean closed time by decreasing both C3 and C2. The cis-unsaturated fatty acid linoleate increased P oalso, whereas the saturated fatty acid myristate and the trans-unsaturated fatty acid elaidate did not affect P o. This channel is activated also by negative pressure applied to the pipette during inside-out recording. Thus we determined the effect of the stretch-activated channel blockers amiloride and Gd3+ on the K+ channel after activation by AA. Amiloride (2 mM) on the extracellular side reduced single-channel amplitude in a voltage-dependent manner, whereas Gd3+ (100 μM) had no effect on channel activity. Activation of this K+ channel may be important during stimulation of Cl− secretion by agonists that use AA as a second messenger (e.g., vasoactive intestinal polypeptide, adenosine) or during the volume regulatory response to cell swelling.

scholarly journals Gating and Inward Rectifying Properties of the MthK K+ Channel with and without the Gating Ring

2007 ◽  
Vol 129 (2) ◽  
pp. 109-120 ◽  
Author(s):  
Yang Li ◽  
Ian Berke ◽  
Liping Chen ◽  
Youxing Jiang
Keyword(s):  
Single Channel ◽  
Hill Coefficient ◽  
K Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Ion Conduction ◽  
Voltage Dependent ◽  
Inwardly Rectifying ◽  
Kinetics Of ◽  
Gating Process

In MthK, a Ca2+-gated K+ channel from Methanobacterium thermoautotrophicum, eight cytoplasmic RCK domains form an octameric gating ring that controls the intracellular gate of the ion conduction pore. The binding of Ca2+ ions to the RCK domains alters the conformation of the gating ring, thereby opening the gate. In the present study, we examined the Ca2+- and pH-regulated gating and the rectifying conduction properties of MthK at the single-channel level. The open probability (Po) of MthK exhibits a sigmoidal relationship with intracellular [Ca2+], and a Hill coefficient &gt;1 is required to describe the dependence of Po on [Ca2+], suggesting cooperative Ca2+ activation of the channel. Additionally, intracellular Ca2+ also blocks the MthK pore in a voltage-dependent manner, rendering an apparently inwardly rectifying I-V relation. Intracellular pH has a dual effect on MthK gating. Below pH 7.5, the channel becomes insensitive to Ca2+. This occurs because the gating ring is structurally unstable at this pH and tends to disassemble (Ye, S., Y. Li, L. Chen, and Y. Jiang. 2006. Cell. 126:1161–1173). In contrast, above pH 7.5, a further increase in pH shifts the Po-[Ca2+] relation towards a lower Ca2+ concentration, augments Po at saturating [Ca2+], and activates the channel even in the absence of Ca2+. Channel activity is marked by bursts of rapid openings and closings separated by relatively longer interburst closings. The duration of interburst closing and the burst length are highly Ca2+ and pH dependent, whereas the kinetics of intraburst events is Ca2+ and pH independent. The rapid intraburst openings and closings are also observed with the isolated MthK pore lacking the attached intracellular gating ring. The fast kinetic events, independent of both Ca2+ and pH, therefore appear to be determined by processes occurring within the ion conduction pore, whereas the slow events reflect the gating process controlled by Ca2+ and pH through the gating ring.

scholarly journals Potentiation of TRPC5 by Protons

2007 ◽  
Vol 282 (46) ◽  
pp. 33868-33878 ◽  
Author(s):  
Marcus Semtner ◽  
Michael Schaefer ◽  
Olaf Pinkenburg ◽  
Tim D. Plant
Keyword(s):  
Phospholipase C ◽  
Transient Receptor Potential ◽  
Single Channel ◽  
Receptor Potential ◽  
High Sensitivity ◽  
Transient Receptor Potential Channel ◽  
Extracellular Ph ◽  
Dependent Manner ◽  
Open Probability ◽  
Transient Receptor

Mammalian members of the classical transient receptor potential channel subfamily (TRPC) are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. TRPC4 and TRPC5 form a group within the TRPC subfamily and are activated in a phospholipase C-dependent manner by an unidentified messenger. Unlike most other Ca2+-permeable channels, TRPC4 and -5 are potentiated by micromolar concentrations of La3+ and Gd3+. This effect results from an action of the cations at two glutamate residues accessible from the extracellular solution. Here, we show that TRPC4 and -5 respond to changes in extracellular pH. Lowering the pH increased both G protein-activated and spontaneous TRPC5 currents. Both effects were already observed with small reductions in pH (from 7.4 to 7.0) and increased up to pH 6.5. TRPC4 was also potentiated by decreases in pH, whereas TRPC6 was only inhibited, with a pIC50 of 5.7. Mutation of the glutamate residues responsible for lanthanoid sensitivity of TRPC5 (E543Q and E595Q) modified the potentiation of TRPC5 by acid. Further evidence for a similarity in the actions of lanthanoids and H+ on TRPC5 is the reduction in single channel conductance and dramatic increase in channel open probability in the presence of either H+ or Gd3+ that leads to larger integral currents. In conclusion, the high sensitivity of TRPC5 to H+ indicates that, in addition to regulation by phospholipase C and other factors, the channel may act as a sensor of pH that links decreases in extracellular pH to Ca2+ entry and depolarization.

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