scholarly journals AKT2/3 Subunits Render Guard Cell K+ Channels Ca2+ Sensitive

2005 ◽  
Vol 125 (5) ◽  
pp. 483-492 ◽  
Author(s):  
Natalya Ivashikina ◽  
Rosalia Deeken ◽  
Susanne Fischer ◽  
Peter Ache ◽  
Rainer Hedrich
Keyword(s):  
Guard Cell ◽  
Guard Cells ◽  
Inward Rectifier ◽  
Hek293 Cells ◽  
K Channel ◽  
Dependent Manner ◽  
Wild Type ◽  
K Channels ◽  
Isolation And Characterization ◽  
Voltage Dependent

Inward-rectifying K+ channels serve as a major pathway for Ca2+-sensitive K+ influx into guard cells. Arabidopsis thaliana guard cell inward-rectifying K+ channels are assembled from multiple K+ channel subunits. Following the recent isolation and characterization of an akt2/3-1 knockout mutant, we examined whether the AKT2/3 subunit carries the Ca2+ sensitivity of the guard cell inward rectifier. Quantification of RT-PCR products showed that despite the absence of AKT2 transcripts in guard cells of the knockout plant, expression levels of the other K+ channel subunits (KAT1, KAT2, AKT1, and AtKC1) remained largely unaffected. Patch-clamp experiments with guard cell protoplasts from wild type and akt2/3-1 mutant, however, revealed pronounced differences in Ca2+ sensitivity of the K+ inward rectifier. Wild-type channels were blocked by extracellular Ca2+ in a concentration- and voltage-dependent manner. Akt2/3-1 mutants lacked the voltage-dependent Ca2+ block, characteristic for the K+ inward rectifier. To confirm the akt2/3-1 phenotype, two independent knockout mutants, akt2-1 and akt2::En-1 were tested, demonstrating that the loss of AKT2/3 indeed affects the Ca2+ dependence of guard cell inward rectifier. In contrast to AKT2 knockout plants, AKT1, AtKC1, and KAT1 loss-of-function mutants retained Ca2+ block of the guard cell inward rectifier. When expressed in HEK293 cells, AKT2 channel displayed a pronounced susceptibility toward extracellular Ca2+, while the dominant guard cell K+ channel KAT2 was Ca2+ insensitive. Thus, we conclude that the AKT2/3 subunit constitutes the Ca2+ sensitivity of the guard cell K+ uptake channel.

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.

scholarly journals Cation permeation through the voltage-dependent potassium channel in the squid axon. Characteristics and mechanisms.

1987 ◽  
Vol 90 (2) ◽  
pp. 261-290 ◽  
Author(s):  
P K Wagoner ◽  
G S Oxford
Keyword(s):  
Binding Energies ◽  
Ion Concentration ◽  
K Channel ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
K Channels ◽  
Energy Profiles ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Ion Binding Sites

Characteristics of cation permeation through voltage-dependent delayed rectifier K channels in squid giant axons were examined. Axial wire voltage-clamp measurements and internal perfusion were used to determine conductance and permeability properties. These K channels exhibit conductance saturation and decline with increases in symmetrical K+ concentrations to 3 M. They also produce ion- and concentration-dependent current-voltage shapes. K channel permeability ratios obtained with substitutions of internal Rb+ or NH+4 for K+ are higher than for external substitution of these ions. Furthermore, conductance and permeability ratios of NH+4 or Rb+ to K+ are functions of ion concentration. Conductance measurements also reveal the presence of an anomalous mole fraction effect for NH+4, Rb+, or Tl+ to K+. Finally, internal Cs+ blocks these K channels in a voltage-dependent manner, with relief of block by elevations in external K+ but not external NH+4 or Cs+. Energy profiles for K+, NH+4, Rb+, Tl+, and Cs+ incorporating three barriers and two ion-binding sites are fitted to the data. The profiles are asymmetric with respect to the center of the electric field, have different binding energies and electrical positions for each ion, and (for K+) exhibit concentration-dependent barrier positions.

scholarly journals Dynamics of aminopyridine block of potassium channels in squid axon membrane.

1976 ◽  
Vol 68 (5) ◽  
pp. 519-535 ◽  
Author(s):  
J Z Yeh ◽  
G S Oxford ◽  
C H Wu ◽  
T Narahashi
Keyword(s):  
Time Course ◽  
Membrane Surface ◽  
K Channel ◽  
Dependent Manner ◽  
Squid Axon ◽  
Axon Membrane ◽  
K Channels ◽  
Voltage Dependent ◽  
K Current ◽  
Kinetics Of

Aminopyridines (2-AP, 3-AP, and 4-AP) selectively block K channels of squid axon membranes in a manner dependent upon the membrane potential and the duration and frequency of voltage clamp pulses. They are effective when applied to either the internal or the external membrane surface. The steady-state block of K channels by aminopyridines is more complete for low depolarizations, and is gradually relieved at higher depolarizations. The K current in the presence of aminopyridines rises more slowly than in control, the change being more conspicuous in 3-AP and 4-AP than in 2-AP. Repetitive pulsing relieves the block in a manner dependent upon the duration and interval of pulses. The recovery from block during a given test pulse is enhanced by increasing the duration of a conditioning depolarizing prepulse. The time constant for this recovery is in the range of 10-20 ms in 3-AP and 4-AP, and shorter in 2-AP. Twin pulse experiments with variable pulse intervals have revealed that the time course for re-establishment of block is much slower in 3-AP and 4-AP than in 2-AP. These results suggest that 2-AP interacts with the K channel more rapidly than 3-AP and 4-AP. The more rapid interaction of 2-AP with K channels is reflected in the kinetics of K current which is faster than that observed in 3-AP or 4-AP, and in the pattern of frequency-dependent block which is different from that in 3-AP or 4-AP. The experimental observations are not satisfactorily described by alterations of Hodgkin-Huxley n-type gating units. Rather, the data are consistent with a simple binding scheme incorporating no changes in gating kinetics which conceives of aminopyridine molecules binding to closed K channels and being released from open channels in a voltage-dependent manner.

A novel approach allows identification of K channels in the lateral membrane of rat CCD

1994 ◽  
Vol 266 (5) ◽  
pp. F813-F822 ◽  
Author(s):  
W. H. Wang ◽  
C. M. McNicholas ◽  
A. S. Segal ◽  
G. Giebisch
Keyword(s):  
Membrane Potential ◽  
Cell Membrane ◽  
Cell Membrane Potential ◽  
K Channel ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Lateral Membrane ◽  
Novel Approach ◽  
Voltage Dependent

We have developed a novel approach to study K channels in the lateral membrane of principal cells (PC) in rat cortical collecting ducts (CCD). The technique consists of 1) exposing the CCD apical membrane, 2) removing the intercalated cells adjoining a PC by gentle suction through a pipette, and 3) applying patch-clamp technique to the lateral membrane of PC. Functional viability of the PC was confirmed by three indexes: 1) maintenance of physiological cell membrane potentials (-85 +/- 3 mV); 2) depolarization of the cell membrane potential with 1 mM Ba2+; and 3) hyperpolarization of the cell potential with 0.1 mM amiloride. Two types of K channels were identified: a low-conductance K channel and an intermediate-conductance K channel. In cell-attached patches the slope conductance of the low-conductance K channel was 27 pS and that of the intermediate-conductance K channel was 45 pS. The open probability (Po) of the 27-pS K channel was 0.81 +/- 0.02 and was not voltage dependent. In contrast, the Po of the 45-pS K channel was 0.23 +/- 0.01 at the spontaneous cell membrane potential and was increased by hyperpolarization. In addition, decrease of the bath pH from 7.4 to 6.7 reduced the 27-pS K channel current amplitude in a voltage-dependent manner, but the Po was not affected. Finally, two time constants were required to fit open- and closed-time histograms of both populations of K channels. Application of 1 mM Ba2+ completely blocked these K channels. We conclude that two types of K channel are present in the basolateral membrane of PC.

Ca-activated K channels in apical membrane of mammalian CCT, and their role in K secretion

1987 ◽  
Vol 252 (3) ◽  
pp. F458-F467 ◽  
Author(s):  
G. Frindt ◽  
L. G. Palmer
Keyword(s):  
Apical Membrane ◽  
K Channel ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Voltage Dependent ◽  
K Secretion ◽  
Excised Patches ◽  
Cytoplasmic Side ◽  
High Conductance

High conductance, Ca-activated K channels were studied in the apical membrane of the rat cortical collecting tubule (CCT) using the patch-clamp technique. In cell-attached patches the channels were found mainly in the closed state at the spontaneous apical membrane potential. They spent progressively more time in the open state as the pipette potential was made negative relative to the bath. In excised patches these channels had a high selectivity for K over Na and were activated by micromolar concentrations of Ca2+ on the cytoplasmic side of the membrane in a voltage-dependent manner. They had a low conductance to Rb and were blocked by Ba (1-100 microM) from the cytoplasmic side and tetraethylammonium (TEA) (0.2-1 mM) from the luminal side. Block by external TEA and small conductance to Rb were used to investigate the role of these channels in K transport by the isolated perfused rabbit CCT. Ba (2.5 mM), a well-studied blocker of apical K conductance in this segment, hyperpolarized the transepithelial voltage (VT) by 3.7 +/- 0.9 mV when added to the luminal solution of the perfused tubule. Addition of TEA (5 mM) to the luminal solution has no effect on VT. When Na transport was abolished by luminal amiloride, perfusion with 30 mM K (replacing Na) resulted in a lumen-negative VT (18-34 mV). Under these conditions, VT was reduced by 6.0 +/- 1.5 mV by 2.5 mM Ba, whereas TEA had no effect. Perfusion with 30 mM Rb (replacing Na) also caused a lumen-negative VT that was approximately 50% of that observed with 30 mM K. The apical K conductance of the perfused CCT appears to be insensitive to luminal TEA and only modestly selective for K over Rb. This conductance, at least under the conditions of our studies, is probably not mediated by the high conductance Ca-activated K channel.

Arabidopsis AtCNGC10 rescues potassium channel mutants of E. coli, yeast and Arabidopsis and is regulated by calcium/calmodulin and cyclic GMP in E. coli

2005 ◽  
Vol 32 (7) ◽  
pp. 643 ◽  
Author(s):  
Xinli Li ◽  
Tamás Borsics ◽  
H. Michael Harrington ◽  
David A. Christopher
Keyword(s):  
Channel Blocker ◽  
K Channel ◽  
Structure Characteristic ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
E Coli ◽  
Diverse Species ◽  
The Family ◽  
Low K

We have isolated and characterised AtCNGC10, one of the 20 members of the family of cyclic nucleotide (CN)-gated and calmodulin (CaM)-regulated channels (CNGCs) from Arabidopsis thaliana (L.) Heynh. AtCNGC10 bound CaM in a C-terminal subregion that contains a basic amphiphillic structure characteristic of CaM-binding proteins and that also overlaps with the predicted CN-binding domain. AtCNGC10 is insensitive to the broad-range K+ channel blocker, tetraethylammonium, and lacks a typical K+-signature motif. However, AtCNGC10 complemented K+ channel uptake mutants of Escherichia coli (LB650), yeast (Saccharomyces cerevisiae CY162) and Arabidopsis (akt1-1). Sense 35S-AtCNGC10 transformed into the Arabidopsis akt1-1 mutant, grew 1.7-fold better on K+-limited medium relative to the vector control. Coexpression of CaM and AtCNGC10 in E. coli showed that Ca2+ / CaM inhibited cell growth by 40%, while cGMP reversed the inhibition by Ca2+ / CaM, in a AtCNGC10-dependent manner. AtCNGC10 did not confer tolerance to Cs+ in E. coli, however, it confers tolerance to toxic levels of Na+ and Cs+ in the yeast K+ uptake mutant grown on low K+ medium. Antisense AtCNGC10 plants had 50% less potassium than wild type Columbia. Taken together, the studies from three evolutionarily diverse species demonstrated a role for the CaM-binding channel, AtCNGC10, in mediating the uptake of K+ in plants.

scholarly journals Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels

2018 ◽  
Vol 115 (47) ◽  
pp. 12051-12056 ◽  
Author(s):  
Akil A. Puckerin ◽  
Donald D. Chang ◽  
Zunaira Shuja ◽  
Papiya Choudhury ◽  
Joachim Scholz ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Channel Blockers ◽  
Wild Type ◽  
Voltage Dependent ◽  
Somatosensory Neurons ◽  
Potential Applications ◽  
Voltage Dependent Calcium Channels ◽  
Potential Therapeutics

Genetically encoded inhibitors for voltage-dependent Ca2+ (CaV) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca2+ (CaV1/CaV2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary CaVβ-subunits. To investigate latent CaVβ-independent components of inhibition, we coexpressed each RGK individually with CaV1 (CaV1.2/CaV1.3) or CaV2 (CaV2.1/CaV2.2) channels reconstituted in HEK293 cells with either wild-type (WT) β2a or a mutant version (β2a,TM) that does not bind RGKs. All four RGKs strongly inhibited CaV1/CaV2 channels reconstituted with WT β2a. By contrast, when channels were reconstituted with β2a,TM, Rem inhibited only CaV1.2, Rad selectively inhibited CaV1.2 and CaV2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT CaVβ, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted CaV1.2 while Rad[R208A/L235A] inhibited CaV1.2/CaV2.2 but not CaV1.3/CaV2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous CaV1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for CaV1.2 and CaV1.2/CaV2.2 channels. Thus, we have exploited latent β-binding–independent Rem and Rad inhibition of specific CaV1/CaV2 channels to develop selective GECCIs with properties unmatched by current small-molecule CaV channel blockers.

Uterine Excitability and Ion Channels and Their Changes with Gestation and Hormonal Environment

2020 ◽  
Vol 83 (1) ◽  
Author(s):  
Susan Wray ◽  
Sarah Arrowsmith
Keyword(s):  
Ion Channels ◽  
Inward Rectifier ◽  
Cation Channels ◽  
Annual Review ◽  
Publication Date ◽  
K Channels ◽  
Voltage Gated ◽  
New Findings ◽  
Voltage Dependent ◽  
Pore Domain

We address advances in the understanding of myometrial physiology, focusing on excitation and the effects of gestation on ion channels and their relevance to labor. This review moves through pioneering studies to exciting new findings. We begin with the myometrium and its myocytes and describe how excitation might initiate and spread in this myogenic smooth muscle. We then review each of the ion channels in the myometrium: L- and T-type Ca2+ channels, KATP (Kir6) channels, voltage-dependent K channels (Kv4, Kv7, and Kv11), twin-pore domain K channels (TASK, TREK), inward rectifier Kir7.1, Ca2+-activated K+ channels with large (KCNMA1, Slo1), small (KCNN1–3), and intermediate (KCNN4) conductance, Na-activated K channels (Slo2), voltage-gated (SCN) Na+ and Na+ leak channels, nonselective (NALCN) channels, the Na K-ATPase, and hyperpolarization-activated cation channels. We finish by assessing how three key hormones— oxytocin, estrogen, and progesterone—modulate and integrate excitability throughout gestation. Expected final online publication date for the Annual Review of Physiology, Volume 83 is February 10, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Rat Prl and TSH secretion are regulated differently by K(+)-channel blockers

1994 ◽  
Vol 266 (1) ◽  
pp. E39-E43 ◽  
Author(s):  
X. Wang ◽  
T. Inukai ◽  
M. A. Greer ◽  
S. E. Greer
Keyword(s):  
Channel Blocker ◽  
Cell Types ◽  
Inhibitory Effect ◽  
Thyroid Stimulating Hormone ◽  
K Channel ◽  
Channel Blockers ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
K Channels ◽  
Tsh Secretion

All four different K(+)-channel blockers [tetraethylammonium (TEA), a nonselective K(+)-channel blocker; tolbutamide, an ATP-sensitive K(+)-channel blocker; quinine and 4-aminopyridine, both primarily voltage-dependent K(+)-channel blockers] stimulated prolactin (Prl) secretion by acutely dispersed anterior pituitary cells but had no effect on thyroid-stimulating hormone (TSH) secretion. TEA stimulated Prl secretion in a dose-dependent manner between 1 microM and 20 mM, but even as high as 20 mM, TEA did not induce TSH secretion. Valinomycin (2 microM), a K+ ionophore, inhibited both basal and TEA-induced Prl secretion. TEA-stimulated Prl secretion was abolished by using a Ca(2+)-depleted medium or adding 10 microM dopamine. TEA did not reverse the inhibitory effect of dopamine on thyrotropin-releasing hormone-induced Prl secretion. Our data indicate that K+ channels may play a role in the secretion of adenohypophysial hormones that is idiosyncratic for each hormone. Differences in the role of K+ channels may reflect differences between the various pituitary cell types in plasma membrane ion channel composition, membrane potential, or the mechanism of exocytosis.

scholarly journals Interaction Mechanisms between Polyamines and IRK1 Inward Rectifier K+ Channels

2003 ◽  
Vol 122 (5) ◽  
pp. 485-500 ◽  
Author(s):  
Donglin Guo ◽  
Zhe Lu
Keyword(s):  
Voltage Dependence ◽  
Divalent Cations ◽  
Inward Rectifier ◽  
Affinity Binding ◽  
Channel Block ◽  
K Channels ◽  
High Affinity ◽  
Voltage Dependent ◽  
Varying Length ◽  
Amine Groups

Rectification of macroscopic current through inward-rectifier K+ (Kir) channels reflects strong voltage dependence of channel block by intracellular cations such as polyamines. The voltage dependence results primarily from the movement of K+ ions across the transmembrane electric field, which accompanies the binding–unbinding of a blocker. Residues D172, E224, and E299 in IRK1 are critical for high-affinity binding of blockers. D172 appears to be located somewhat internal to the narrow K+ selectivity filter, whereas E224 and E299 form a ring at a more intracellular site. Using a series of alkyl-bis-amines of varying length as calibration, we investigated how the acidic residues in IRK1 interact with amine groups in the natural polyamines (putrescine, spermidine, and spermine) that cause rectification in cells. To block the pore, the leading amine of bis-amines of increasing length penetrates ever deeper into the pore toward D172, while the trailing amine in every bis-amine binds near a more intracellular site and interacts with E224 and E299. The leading amine in nonamethylene-bis-amine (bis-C9) makes the closest approach to D172, displacing the maximal number of K+ ions and exhibiting the strongest voltage dependence. Cells do not synthesize bis-amines longer than putrescine (bis-C4) but generate the polyamines spermidine and spermine by attaching an amino-propyl group to one or both ends of putrescine. Voltage dependence of channel block by the tetra-amine spermine is comparable to that of block by the bis-amines bis-C9 (shorter) or bis-C12 (equally long), but spermine binds to IRK1 with much higher affinity than either bis-amine does. Thus, counterintuitively, the multiple amines in spermine primarily confer the high affinity but not the strong voltage dependence of channel block. Tetravalent spermine achieves a stronger interaction with the pore by effectively behaving like a pair of tethered divalent cations, two amine groups in its leading half interacting primarily with D172, whereas the other two in the trailing half interact primarily with E224 and E299. Thus, nature has optimized not only the blocker but also, in a complementary manner, the channel for producing rapid, high-affinity, and strongly voltage-dependent channel block, giving rise to exceedingly sharp rectification.

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