A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K⁺ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K⁺ currents, an inactivating outward-rectifying K⁺ current was characterized (plant A-type current: I_AP). I_AP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. I_AP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to −100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward I_AP was mainly carried by K⁺ ions. In contrast to the sustained outward-rectifying K⁺ currents, cytosolic alkaline pH was found to inhibit I_AP and extracellular K⁺ was required for I_AP activity. Furthermore, increasing cytosolic free Ca²⁺ in the physiological range strongly inhibited I_AP activity with a half inhibitory concentration of ≈ 94 nM. We present a detailed characterization of an inactivating K⁺ current in a higher plant cell. Regulation of I_AP by diverse factors including membrane potential, cytosolic Ca²⁺ and pH, and extracellular K⁺ and Ca²⁺ implies that the inactivating I_AP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.     

Determination of transmembrane topology of an inward-rectifying potassium channel from Arabidopsis thaliana based on functional expression in Escherichia coli

We report here that the inward-rectifying potassium channels KAT1 and AKT2 were functionally expressed in K⁺ uptake-deficient Escherichia coli. Immunological assays showed that KAT1 was translocated into the cell membrane of E. coli. Functional assays suggested that KAT1 was inserted topologically correctly into the cell membrane. In control experiments, the inactive point mutation in KAT1, T256R, did not complement for K⁺ uptake in E. coli. The inward-rectifying K⁺ channels of plants share a common hydrophobic domain comprising at least six membrane-spanning segments (S1–S6). The finding that a K⁺ channel can be expressed in bacteria was further exploited to determine the KAT1 membrane topology by a gene fusion approach using the bacterial reporter enzymes, alkaline phosphatase, which is active only in the periplasm, and β-galactosidase. The enzyme activity from the alkaline phosphatase and β-galactosidase fusion plasmid showed that the widely predicted S1, S2, S5, and S6 segments were inserted into the membrane. Although the S3 segment in the alkaline phosphatase fusion protein could not function as an export signal, the replacement of a negatively charged residue inside S3 with a neutral amino acid resulted in an increase in alkaline phosphatase activity, which indicates that the alkaline phosphatase was translocated into the periplasm. For membrane translocation of S3, the neutralization of a negatively charged residue in S3 may be required presumably because of pairing with a positively charged residue of S4. These results revealed that KAT1 has the common six transmembrane-spanning membrane topology that has been predicted for the Shaker superfamily of voltage-dependent K⁺ channels. Furthermore, the functional complementation of a bacterial K⁺ uptake mutant in this study is shown to be an alternative expression system for plant K⁺ channel proteins and a potent tool for their topological analysis.     

Regulation of the human ether-a-gogo related gene (HERG) K+ channels by reactive oxygen species

Human ether-a-gogo related gene (HERG) K⁺ channels are key elements in the control of cell excitability in both the cardiovascular and the central nervous systems. For this reason, the possible modulation by reactive oxygen species (ROS) of HERG and other cloned K⁺ channels expressed in Xenopus oocytes has been explored in the present study. Exposure of Xenopus oocytes to an extracellular solution containing FeSO₄ (25–100 μM) and ascorbic acid (50–200 μM) (Fe/Asc) increased both malondialdehyde content and 2′,7′-dichlorofluorescin fluorescence, two indexes of ROS production. Oocyte perfusion with Fe/Asc caused a 50% increase of the outward K⁺ currents carried by HERG channels, whereas inward currents were not modified. This ROS-induced increase in HERG outward K⁺ currents was due to a depolarizing shift of the voltage-dependence of channel inactivation, with no change in channel activation. No effect of Fe/Asc was observed on the expressed K⁺ currents carried by other K⁺ channels such as bEAG, rDRK1, and mIRK1. Fe/Asc-induced stimulation of HERG outward currents was completely prevented by perfusion of the oocytes with a ROS scavenger mixture (containing 1,000 units/ml catalase, 200 ng/ml superoxide dismutase, and 2 mM mannitol). Furthermore, the scavenger mixture also was able to reduce HERG outward currents in resting conditions by 30%, an effect mimicked by catalase alone. In conclusion, the present results seem to suggest that changes in ROS production can specifically influence K⁺ currents carried by the HERG channels.     

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo)

Large conductance calcium- and voltage-sensitive K⁺ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca²⁺ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca²⁺ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4–5 elementary charges.     

Gating modifier toxins reveal a conserved structural motif in voltage-gated Ca2+ and K+ channels

Protein toxins from venomous animals exhibit remarkably specific and selective interactions with a wide variety of ion channels. Hanatoxin and grammotoxin are two related protein toxins found in the venom of the Chilean Rose Tarantula, Phrixotrichus spatulata. Hanatoxin inhibits voltage-gated K⁺ channels and grammotoxin inhibits voltage-gated Ca²⁺ channels. Both toxins inhibit their respective channels by interfering with normal operation of the voltage-dependent gating mechanism. The sequence homology of hanatoxin and grammotoxin, as well as their similar mechanism of action, raises the possibility that they interact with the same region of voltage-gated Ca²⁺ and K⁺ channels. Here, we show that each toxin can interact with both voltage-gated Ca²⁺ and K⁺ channels and modify channel gating. Moreover, mutagenesis of voltage-gated K⁺ channels suggests that hanatoxin and grammotoxin recognize the same structural motif. We propose that these toxins recognize a voltage-sensing domain or module present in voltage-gated ion channels and that this domain has a highly conserved three-dimensional structure.     

Killing K Channels with TEA+

Tetraethylammonium (TEA⁺) is widely used for reversible blockade of K channels in many preparations. We noticed that intracellular perfusion of voltage-clamped squid giant axons with a solution containing K⁺ and TEA⁺ irreversibly decreased the potassium current when there was no K⁺ outside. Five minutes of perfusion with 20 mM TEA⁺, followed by removal of TEA⁺, reduced potassium current to <5% of its initial value. The irreversible disappearance of K channels with TEA⁺ could be prevented by addition of ≥ 10 mM K⁺ to the extracellular solution. The rate of disappearance of K channels followed first-order kinetics and was slowed by reducing the concentration of TEA⁺. Killing is much less evident when an axon is held at −110 mV to tightly close all of the channels. The longer-chain TEA⁺ derivative decyltriethylammonium (C10⁺) had irreversible effects similar to TEA⁺. External K⁺ also protected K channels against the irreversible action of C10⁺. It has been reported that removal of all K⁺ internally and externally (dekalification) can result in the disappearance of K channels, suggesting that binding of K⁺ within the pore is required to maintain function. Our evidence further suggests that the crucial location for K⁺ binding is external to the (internal) TEA⁺ site and that TEA⁺ prevents refilling of this location by intracellular K⁺. Thus in the absence of extracellular K⁺, application of TEA⁺ (or C10⁺) has effects resembling dekalification and kills the K channels.     

ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae

A K⁺ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K⁺ uptake. Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K⁺ transport capacity on yeast cells when heterologously expressed. In Xenopus laevis oocytes, expression yields an ungated K⁺-selective current whose attributes resemble the “leak” conductance thought to mediate the resting potential of vertebrate myelinated neurons but whose molecular nature has long remained elusive. The predicted protein has two pore (P) domains and four membrane-spanning helices and is a member of a newly recognized K⁺ channel family. Expression of the channel in flies and yeast cells makes feasible studies of structure and in vivo function using genetic approaches that are not possible in higher animals.     

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H+ and Na+(K+)/H+ exchange

Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na⁺- and K⁺-dependent pH homeostasis and in Na⁺ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)–hexahistidine proteoliposomes exhibit high activities of tetracycline–cobalt/H⁺, Na⁺/H⁺, and K⁺/H⁺ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline–cobalt/H⁺ and Na⁺/H⁺ antiports is presented. K⁺ and Li⁺ inhibit Na⁺ uptake, but there is little cross-inhibition between Na⁺ and tetracycline–cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.     

A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K⁺ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by G_βγ subunits, but instead shows constitutive activation, and (ii) is no longer a K⁺-selective channel but conducts Na⁺ as well. The present experiments on weaverGIRK2 (wvGIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na⁺ concentration. In particular, manipulations that decrease intracellular Na⁺ produce a component of Na⁺-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na⁺ influx through the wvGIRK2 channel, leading to additional Na⁺ activation. We also show that the wvGIRK2 channel is permeable to Ca²⁺, providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K⁺ channels.     

Long QT and ventricular arrhythmias in transgenic mice expressing the N terminus and first transmembrane segment of a voltage-gated potassium channel

Voltage-gated potassium channels control cardiac repolarization, and mutations of K⁺ channel genes recently have been shown to cause arrhythmias and sudden death in families with the congenital long QT syndrome. The precise mechanism by which the mutations lead to QT prolongation and arrhythmias is uncertain, however. We have shown previously that an N-terminal fragment including the first transmembrane segment of the rat delayed rectifier K⁺ channel Kv1.1 (Kv1.1N206Tag) coassembles with other K⁺ channels of the Kv1 subfamily in vitro, inhibits the currents encoded by Kv1.5 in a dominant-negative manner when coexpressed in Xenopus oocytes, and traps Kv1.5 polypeptide in the endoplasmic reticulum of GH3 cells. Here we report that transgenic mice overexpressing Kv1.1N206Tag in the heart have a prolonged QT interval and ventricular tachycardia. Cardiac myocytes from these mice have action potential prolongation caused by a significant reduction in the density of a rapidly activating, slowly inactivating, 4-aminopyridine sensitive outward K⁺ current. These changes correlate with a marked decrease in the level of Kv1.5 polypeptide. Thus, overexpression of a truncated K⁺ channel in the heart alters native K⁺ channel expression and has profound effects on cardiacexcitability.     

The selectivity of the hair cell’s mechanoelectrical-transduction channel promotes Ca2+ flux at low Ca2+ concentrations

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca²⁺ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel’s open probability, an understanding of adaptation requires estimation of the magnitude of Ca²⁺ influx. To determine the Ca²⁺ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K⁺, Na⁺, or Ca²⁺. For modest concentrations of a single permeant cation, Ca²⁺ carried much more receptor current than did either K⁺ or Na⁺. For higher cation concentrations, however, the flux of Na⁺ or K⁺ through the transduction channel exceeded that of Ca²⁺. For mixtures of Ca²⁺ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel’s pore. These results demonstrate not only that the hair cell’s transduction channel is selective for Ca²⁺ over monovalent cations but also that Ca²⁺ carries substantial current even at low Ca²⁺ concentrations. At physiological cation concentrations, Ca²⁺ flux through transduction channels can change the local Ca²⁺ concentration in stereocilia in a range relevant for the control of adaptation.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Defective γ-aminobutyric acid type B receptor-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from weaver and Girk2 null mutant mice

Stimulation of inhibitory neurotransmitter receptors, such as γ-aminobutyric acid type B (GABA_B) receptors, activates G protein-gated inwardly rectifying K⁺ channels (GIRK) which, in turn, influence membrane excitability. Seizure activity has been reported in a Girk2 null mutant mouse lacking GIRK2 channels but showing normal cerebellar development as well as in the weaver mouse, which has mutated GIRK2 channels and shows abnormal development. To understand how the function of GIRK2 channels differs in these two mutant mice, we compared the G protein-activated inwardly rectifying K⁺ currents in cerebellar granule cells isolated from Girk2 null mutant and weaver mutant mice with those from wild-type mice. Activation of GABA_B receptors in wild-type granule cells induced an inwardly rectifying K⁺ current, which was sensitive to pertussis toxin and inhibited by external Ba²⁺ ions. The amplitude of the GABA_B receptor-activated current was severely attenuated in granule cells isolated from both weaver and Girk2 null mutant mice. By contrast, the G protein-gated inwardly rectifying current and possibly the agonist-independent basal current appeared to be less selective for K⁺ ions in weaver but not Girk2 null mutant granule cells. Our results support the hypothesis that a nonselective current leads to the weaver phenotype. The loss of GABA_B receptor-activated GIRK current appears coincident with the absence of GIRK2 channel protein and the reduction of GIRK1 channel protein in the Girk2 null mutant mouse, suggesting that GABA_B receptors couple to heteromultimers composed of GIRK1 and GIRK2 channel subunits.     

Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-X_L monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at ≈pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl⁻ selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl⁻-selective (P_K/P_Cl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K⁺-selective (P_K/P_Cl = 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K⁺ selectivity (P_K/P_Cl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.     

Normal cerebellar development but susceptibility to seizures in mice lacking G protein-coupled, inwardly rectifying K+ channel GIRK2

G protein-gated, inwardly rectifying K⁺ channels (GIRK) are effectors of G protein-coupled receptors for neurotransmitters and hormones and may play an important role in the regulation of neuronal excitability. GIRK channels may be important in neurodevelopment, as suggested by the recent finding that a point mutation in the pore region of GIRK2 (G156S) is responsible for the weaver (wv) phenotype. The GIRK2 G156S gene gives rise to channels that exhibit a loss of K⁺ selectivity and may also exert dominant-negative effects on G_βγ-activated K⁺ currents. To investigate the physiological role of GIRK2, we generated mutant mice lacking GIRK2. Unlike wv/wv mutant mice, GIRK2 −/− mice are morphologically indistinguishable from wild-type mice, suggesting that the wv phenotype is likely due to abnormal GIRK2 function. Like wv/wv mice, GIRK2 −/− mice have much reduced GIRK1 expression in the brain. They also develop spontaneous seizures and are more susceptible to pharmacologically induced seizures using a γ-aminobutyric acid antagonist. Moreover, wv/− mice exhibit much milder cerebellar abnormalities than wv/wv mice, indicating a dosage effect of the GIRK2 G156S mutation. Our results indicate that the weaver phenotypes arise from a gain-of-function mutation of GIRK2 and that GIRK1 and GIRK2 are important mediators of neuronal excitability in vivo.     

Activation of the atrial KACh channel by the βγ subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates

The βγ subunits of GTP-binding proteins (G_βγ) activate the muscarinic K⁺ channel (K_ACh) in heart by direct binding to both of its component subunits. K_ACh channels can also be gated by internal Na⁺ ions. Both activation mechanisms show dependence on hydrolysis of intracellular ATP. We report that phosphatidylinositol 4,5-bisphosphate (PIP₂) mimics the ATP effects and that depletion or block of PIP₂ retards the stimulatory effects of G_βγ subunits or Na⁺ ions on channel activity, effects that can be reversed by restoring PIP₂. Thus, regulation of K_ACh channel activity may be crucially dependent on PIP₂ and phosphatidylinositol signaling. These striking functional results are in agreement with in vitro biochemical studies on the PIP₂ requirement for G_βγ stimulation of G protein receptor kinase activity, thus implicating phosphatidylinositol phospholipids as a potential control point for G_βγ-mediated signal transduction.     

An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance

Potassium (K⁺) nutrition and salt tolerance are key factors controlling plant productivity. However, the mechanisms by which plants regulate K⁺ nutrition and salt tolerance are poorly understood. We report here the identification of an Arabidopsis thaliana mutant, sos3 (salt-overly-sensitive 3), which is hypersensitive to Na⁺ and Li⁺ stresses. The mutation is recessive and is in a nuclear gene that maps to chromosome V. The sos3 mutation also renders the plant unable to grow on low K⁺. Surprisingly, increased extracellular Ca²⁺ suppresses the growth defect of sos3 plants on low K⁺ or 50 mM NaCl. In contrast, high concentrations of external Ca²⁺ do not rescue the growth of the salt-hypersensitive sos1 mutant on low K⁺ or 50 mM NaCl. Under NaCl stress, sos3 seedlings accumulated more Na⁺ and less K⁺ than the wild type. Increased external Ca²⁺ improved K⁺/Na⁺ selectivity of both sos3 and wild-type plants. However, this Ca²⁺ effect in sos3 is more than twice as much as that in the wild type. In addition to defining the first plant mutant with an altered calcium response, these results demonstrate that the SOS3 locus is essential for K⁺ nutrition, K⁺/Na⁺ selectivity, and salt tolerance in higher plants.     

Stabilization of ion selectivity filter by pore loop ion pairs in an inwardly rectifying potassium channel

Ion selectivity is critical for the biological functions of voltage-dependent cation channels and is achieved by specific ion binding to a pore region called the selectivity filter. In voltage-gated K⁺, Na⁺ and Ca²⁺ channels, the selectivity filter is formed by a short polypeptide loop (called the H5 or P region) between the fifth and sixth transmembrane segments, donated by each of the four subunits or internal homologous domains forming the channel. While mutagenesis studies on voltage-gated K⁺ channels have begun to shed light on the structural organization of this pore region, little is known about the physical and chemical interactions that maintain the structural stability of the selectivity filter. Here we show that in an inwardly rectifying K⁺ (IRK) channel, IRK1, short range interactions of an ion pair in the H5 pore loop are crucial for pore structure and ion permeation. The two residues, a glutamate and an arginine, appear to form exposed salt bridges in the tetrameric channel. Alteration or disruption of such ion pair interactions dramatically alters ion selectivity and permeation. Since this ion pair is conserved in all IRK channels, it may constitute a general mechanism for maintaining the stability of the pore structure in this channel superfamily.     

Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone

We have used Arabidopsis calmodulin (CaM) covalently coupled to horseradish peroxidase to screen a barley aleurone cDNA expression library for CaM binding proteins. The deduced amino acid sequence of one cDNA obtained by this screen was shown to be a unique protein of 702 amino acids with CaM and cyclic nucleotide binding domains at the carboxyl terminus and high similarity to olfactory and K⁺ channels. This cDNA was designated HvCBT1 (Hordeum vulgare CaM binding transporter). Hydropathy plots of HvCBT1 showed the presence of six putative transmembrane domains, but sequence alignment indicated a pore domain that was unlike the consensus domains in K⁺ and olfactory channels. Expression of a subclone of amino acids 482–702 in Escherichia coli generated a peptide that bound CaM. When a fusion protein of HvCBT1 and green fluorescent protein was expressed in barley aleurone protoplasts, fluorescence accumulated in the plasma membrane. Expression of HvCBT1 in the K⁺ transport deficient Saccharomyces cerevisiae mutant CY162 showed no rescue of the mutant phenotype. However, growth of CY162 expressing HvCBT1 with its pore mutated to GYGD, the consensus sequence of K⁺ channels, was compromised. We interpret these data as indicating that HvCBT1 acts to interfere with ion transport.     

Cloning and characterization of a potassium-coupled amino acid transporter

Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na⁺ or H⁺ gradients. The present work extends the Na⁺ and H⁺ dogma by including the H⁺ and K⁺ paradigm. Lepidopteran insect larvae have a high K⁺ and a low Na⁺ content, and their midgut cells lack Na⁺/K⁺ ATPase. Instead, an H⁺ translocating, vacuolar-type ATPase generates a voltage of approximately −240 mV across the apical plasma membrane of so-called goblet cells, which drives H⁺ back into the cells in exchange for K⁺, resulting in net K⁺ secretion into the lumen. The resulting inwardly directed K⁺ electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells. By using expression cloning with Xenopus laevis oocytes, we have isolated a cDNA that encodes a K⁺-coupled amino acid transporter (KAAT1). We have cloned this protein from a larval lepidopteran midgut (Manduca sexta) cDNA library. KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands. When expressed in Xenopus oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes α-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B. K⁺, Na⁺, and to a lesser extent Li⁺ were accepted as cotransported ions, but K⁺ is the principal cation, by far, in living caterpillars. Moreover, uptake was Cl⁻-dependent, and the K⁺/Na⁺ selectivity increased with hyperpolarization of oocytes, reflecting the increased K⁺/Na⁺ selectivity with hyperpolarization observed in midgut tissue. KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na⁺ and Cl⁻-coupled neurotransmitter transporter family.     

Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus

Large conductance voltage- and Ca²⁺-dependent K⁺ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers β subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and “in vivo” reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.