Electrophysiological properties of NtTPK1 expressed in yeast tonoplast

The patch clamp technique using enlarged yeast tonoplasts is an effective approach to characterizing the Nicotiana tabacum plant tonoplast K+ channel, NtTPK1. We report here that the NtTPK1-mediated currents comprise two phase components, both which were found to be highly selective for K+ over Na+ and Cl-.     

A type of voltage-dependent Ca2+ channel on Vicia faba guard cell plasma membrane outwardly permeates K+

The fine regulation of stomatal aperture is important for both plant photosynthesis and transpiration, while stomatal closing is an essential plant response to biotic and abiotic stresses such as drought, salinity, wounding, and pathogens. Quick stomatal closing is primarily due to rapid solute loss. Cytosolic free calcium ([Ca(2+)](cyt)) is a ubiquitous second messenger, and its elevation or oscillation plays important roles in stomatal movements, which can be triggered by the opening of Ca(2+)-permeable channels on the plasma membrane. For Ca(2+)-permeable channel recordings, Ba(2+) is preferred as a charge-carrying ion because it has higher permeability to Ca(2+) channels and blocks K(+) channel activities to facilitate current recordings; however, it prevents visualization of Ca(2+) channels' K(+) permeability. Here, we employed Ca(2+) instead of Ba(2+) in recording Ca(2+)-permeable channels on Vicia faba guard cell plasma membrane to mimic physiological solute conditions inside guard cells more accurately. Inward Ca(2+) currents could be recorded at the single-channel level, and these currents could be inhibited by micromolar Gd(3+), but their reversal potential is far away from the theoretical equilibrium potential for Ca(2+). Further experiments showed that the discrepancy of the reversal potential of the recorded Ca(2+) currents is influenced by cytosolic K(+). This suggests that voltage-dependent Ca(2+) channels also mediate K(+) efflux at depolarization voltages. In addition, a new kind of high-conductance channels with fivefold to normal Ca(2+) channel and 18-fold to normal outward K(+) conductance was found. Our data presented here suggest that plants have their own saving strategies in their rapid response to stress stimuli, and multiple kinds of hyperpolarization-activated Ca(2+)-permeable channels coexist on plasma membranes.     

Role of 20-HETE in the hypoxia-induced activation of Ca2+-activated K+ channel currents in rat cerebral arterial muscle cells

The mechanism of sensing hypoxia and hypoxia-induced activation of cerebral arterial Ca(2+)-activated K(+) (K(Ca)) channel currents and vasodilation is not known. We investigated the roles of the cytochrome P-450 4A (CYP 4A) omega-hydroxylase metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), and generation of superoxide in the hypoxia-evoked activation of the K(Ca) channel current in rat cerebral arterial muscle cells (CAMCs) and cerebral vasodilation. Patch-clamp analysis of K(+) channel current identified a voltage- and Ca(2+)-dependent 238 +/- 21-pS unitary K(+) currents that are inhibitable by tetraethylammonium (TEA, 1 mM) or iberiotoxin (100 nM). Hypoxia (<2% O(2)) reversibly enhanced the open-state probability (NP(o)) of the 238-pS unitary K(Ca) current in cell-attached patches. This effect of hypoxia was not observed on unitary K(Ca) currents recorded from either excised inside-out or outside-out membrane patches. Inhibition of CYP 4A omega-hydroxylase activity increased the NP(o) of K(Ca) single-channel current. Hypoxia reduced the basal endogenous level of 20-HETE by 47 +/- 3% as well as catalytic formation of 20-HETE in cerebral arterial muscle homogenates as determined by liquid chromatography-mass spectrometry analysis. The concentration of authentic 20-HETE was reduced when incubated with the superoxide donor KO(2). Exogenous 20-HETE (100 nM) attenuated the hypoxia-induced activation of the K(Ca) current in CAMCs. Hypoxia did not augment the increase in NP(o) of K(Ca) channel current induced by suicide inhibition of endogenous CYP 4A omega-hydroxylase activity with 17-octadecynoic acid. In pressure (80 mmHg)-constricted cerebral arterial segments, hypoxia induced dilation that was partly attenuated by 20-HETE or by the K(Ca) channel blocker TEA. Exposure to hypoxia caused the generation of intracellular superoxide as evidenced by intense staining of arterial muscle with the fluorescent probe hydroethidine, by quantitation using fluorescent HPLC analysis, and by attenuation of the hypoxia-induced activation of the K(Ca) channel current by superoxide dismutation. These results suggest that the exposure of CAMCs to hypoxia results in the generation of superoxide and reduction in endogenous level of 20-HETE that may account for the hypoxia-induced activation of arterial K(Ca) channel currents and cerebral vasodilation.     

RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte expression studies

The physiological properties of most ion channels are defined experimentally by functional expression of their pore-forming alpha subunits in Xenopus laevis oocytes. Here, we cloned a family of Xenopus KCNE genes that encode MinK-related peptide K(+) channel beta subunits (xMiRPs) and demonstrated their constitutive expression in oocytes. Electrophysiological analysis of xMiRP2 revealed that when overexpressed this gene modulates human cardiac K(+) channel alpha subunits HERG (human ether-a-go-go-related gene) and KCNQ1 by suppressing HERG currents and removing the voltage dependence of KCNQ1 activation. The ability of endogenous levels of xMiRP2 to contribute to the biophysical attributes of overexpressed mammalian K(+) channels in oocyte studies was assessed next. Injection of an xMiRP2 sequence-specific short interfering RNA (siRNA) oligo reduced endogenous xMiRP2 expression 5-fold, whereas a control siRNA oligo had no effect, indicating the effectiveness of the RNA interference technique in Xenopus oocytes. The functional effects of endogenous xMiRP2 silencing were tested using electrophysiological analysis of heterologously expressed HERG channels. The RNA interference-mediated reduction of endogenous xMiRP2 expression increased macroscopic HERG current as much as 10-fold depending on HERG cRNA concentration. The functional effects of human MiRP1 (hMiRP1)/HERG interaction were also affected by endogenous xMiRP2. At high HERG channel density, at which the effects of endogenous xMiRP2 are minimal, hMiRP1 reduced HERG current. At low HERG current density, hMiRP1 paradoxically up-regulated HERG current, a result consistent with hMiRP1 rescuing HERG from suppression by endogenous xMiRP2. Thus, endogenous Xenopus MiRP subunits contribute to the base-line properties of K(+) channels like HERG in oocyte expression studies, which could explain expression level- and expression system-dependent variation in K(+) channel function.     

Selective mobility and sensitivity to SNAREs is exhibited by the Arabidopsis KAT1 K+ channel at the plasma membrane

Recent findings indicate that proteins in the SNARE superfamily are essential for cell signaling, in addition to facilitating vesicle traffic in plant cell homeostasis, growth, and development. We previously identified SNAREs SYP121/Syr1 from tobacco (Nicotiana tabacum) and the Arabidopsis thaliana homolog SYP121 associated with abscisic acid and drought stress. Disrupting tobacco SYP121 function by expressing a dominant-negative Sp2 fragment had severe effects on growth, development, and traffic to the plasma membrane, and it blocked K(+) and Cl(-) channel responses to abscisic acid in guard cells. These observations raise questions about SNARE control in exocytosis and endocytosis of ion channel proteins and their organization within the plane of the membrane. We have used a dual, in vivo tagging strategy with a photoactivatable green fluorescent protein and externally exposed hemagglutinin epitopes to monitor the distribution and trafficking dynamics of the KAT1 K(+) channel transiently expressed in tobacco leaves. KAT1 is localized to the plasma membrane within positionally stable microdomains of approximately 0.5 microm in diameter; delivery of the K(+) channel, but not of the PMA2 H(+)-ATPase, to the plasma membrane is suppressed by Sp2 fragments of tobacco and Arabidopsis SYP121, and Sp2 expression leads to profound changes in KAT1 distribution and mobility within the plane of the plasma membrane. These results offer direct evidence for SNARE-mediated traffic of the K(+) channel and a role in its distribution within subdomains of the plasma membrane, and they implicate a role for SNAREs in positional anchoring of the K(+) channel protein.     

Ca2(+)-activated K+ channels from rabbit kidney medullary thick ascending limb cells expressed in Xenopus oocytes.

Ca2(+)-activated K+ channels are present in muscle, nerve, pancreas, macrophages, and renal cells. They are important in such diverse functions as neurotransmitter release, muscle excitability, pancreatic secretion, and cell volume regulation. Although much is known about the biophysics of Ca2(+)-activated K+ channels, the molecular structure, cDNA and amino acid sequences are unknown. We injected size-fractionated mRNA isolated from cultured rabbit kidney medullary thick ascending limb cells in Xenopus oocytes and observed newly expressed K+ currents using two-microelectrode voltage-clamp technique. The expressed K+ currents are Ca2+ dependent and inhibited by charybdotoxin, a specific blocker of Ca2(+)-activated K+ channels. Amplitudes of the current ranged from 30 nA to more than 1 microA at a membrane potential of +30 mV. Reversal potential of the current suggested a K(+)-selective channel. The peak activity of Ca2(+)-activated K+ channels were observed in fractions corresponding to a message RNA with size of approximately 4.5 kilobases.     

The poplar K+ channel KPT1 is associated with K+ uptake during stomatal opening and bud development

To gain insights into the performance of poplar guard cells, we have measured stomatal conductance and aperture, guard cell K+ content and K+-channel activity of the guard cell plasma membrane in intact poplar leaves. In contrast to Arabidopsis, broad bean and tobacco grown under same conditions, poplar stomata operated just in the dynamic range - any change in conductance altered the rate of photosynthesis. In response to light, CO2 and abscisic acid (ABA), the stomatal opening velocity was two to five times faster than that measured for Arabidopsis thaliana, Nicotiana tabacum and Vicia faba. When stomata opened, the K+ content of guard cells increased almost twofold, indicating that the very fast stomatal opening in this species is mediated via potassium uptake. Following impalement of single guard cells embedded in their natural environment of intact leaves with triple-barrelled microelectrodes, time-dependent inward and outward-rectifying K+-channel-mediated currents of large amplitude were recorded. To analyse the molecular nature of genes encoding guard cell K+-uptake channels, we cloned K+-transporter Populustremula (KPT)1 and functionally expressed this potassium channel in a K+-uptake-deficient Escherichia coli mutant. In addition to guard cells, this K+-transporter gene was expressed in buds, where the KPT1 gene activity strongly correlated with bud break. Thus, KPT1 represents one of only few poplar genes associated with bud flush.     

Intramembrane Charge Movement Associated with Endogenous K+ Channel Activity in HEK-293 Cells

1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.     

Characterization of a chloroplast inner envelope K+ channel.

A K(+)-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as Mg2+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope.     

BmBKTx1, a novel Ca2+-activated K+ channel blocker purified from the Asian scorpion Buthus martensi Karsch

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.     

Blockade of cardiac sarcoplasmic reticulum K+ channel by Ca2+: two-binding-site model of blockade.

Potassium countercurrent through the SR K+ channel plays an important role in Ca2+ release from the SR. To see if Ca2+ regulates the channel, we incorporated canine cardiac SR K+ channel into lipid bilayers. Calcium ions present in either the SR lumenal (trans) or cytoplasmic (cis) side blocked the cardiac SR K+ channel in a voltage-dependent manner. When Ca2+ was present on both sides, however, the block appeared to be voltage independent. A two-binding site model of blockade by an impermeant divalent cation (Ca2+) can explain this apparent contradiction. Estimates of SR Ca2+ concentration suggest that under physiological conditions the cardiac SR K+ channel is partially blocked by Ca2+ ions present in the lumen of the SR. The reduction in lumenal [Ca2+] during Ca2+ release could increase K+ conductance.     

Blunted IgE-mediated activation of mast cells in mice lacking the Ca2+-activated K+ channel KCa3.1

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.     

TWIK-2, an inactivating 2P domain K+ channel

We cloned human and rat TWIK-2 and expressed this novel 2P domain K(+) channel in transiently transfected COS cells. TWIK-2 is highly expressed in the gastrointestinal tract, the vasculature, and the immune system. Rat TWIK-2 currents are about 15 times larger than human TWIK-2 currents, but both exhibit outward rectification in a physiological K(+) gradient and mild inward rectification in symmetrical K(+) conditions. TWIK-2 currents are inactivating at depolarized potentials, and the kinetic of inactivation is highly temperature-sensitive. TWIK-2 shows an extremely low conductance, which prevents the visualization of discrete single channel events. The inactivation and rectification are intrinsic properties of TWIK-2 channels. In a physiological K(+) gradient, TWIK-2 is half inhibited by 0.1 mm Ba(2+), quinine, and quinidine. Finally, cysteine 53 in the M1P1 external loop is required for functional expression of TWIK-2 but is not critical for subunit self-assembly. TWIK-2 is the first reported 2P domain K(+) channel that inactivates. The base-line, transient, and delayed activities of TWIK-2 suggest that this novel 2P domain K(+) channel may play an important functional role in cell electrogenesis.     

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.