K+ activation of kir3.1/kir3.4 and kv1.4 K+ channels is regulated by extracellular charges

K+ activates many inward rectifier and voltage-gated K+ channels. In each case, an increase in K+ current through the channel can occur despite a reduced driving force. We have investigated the molecular mechanism of K+ activation of the inward rectifier K+ channel, Kir3.1/Kir3.4, and the voltage-gated K+ channel, Kv1.4. In the Kir3.1/Kir3.4 channel, mutation of an extracellular arginine residue, R155, in the Kir3.4 subunit markedly reduced K+ activation of the channel. The same mutation also abolished Mg2+ block of the channel. Mutation of the equivalent residue in Kv1.4 (K532) abolished K+ activation as well as C-type inactivation of the Kv1.4 channel. Thus, whereas C-type inactivation is a collapse of the selectivity filter, K+ activation could be an opening of the selectivity filter. K+ activation of the Kv1.4 channel was enhanced by acidic pH. Mutation of an extracellular histidine residue, H508, that mediates the inhibitory effect of protons on Kv1.4 current, abolished both K+ activation and the enhancement of K+ activation at acidic pH. These results suggest that the extracellular positive charges in both the Kir3.1/Kir3.4 and the Kv1.4 channels act as "guards" and regulate access of K+ to the selectivity filter and, thus, the open probability of the selectivity filter. Furthermore, these data suggest that, at acidic pH, protonation of H508 inhibits current through the Kv1.4 channel by decreasing K+ access to the selectivity filter, thus favoring the collapse of the selectivity filter.     

Neuronal transmission stimulates the phosphorylation of Kv1.4 channel at Ser229 through protein kinase A1

Phosphorylation of voltage-gated K+ channels (Kv) is involved in regulation of neuronal excitability, synaptic plasticity and neuronal survival. Among Kv channels expressed in the CNS, Kv1.4 is located in the soma, dendrite and axon terminus of neurones in most regions of the brain. Here, we show that Ser229 found within the highly conserved T1 domain of Kv1.4 in cultured rat cortical neurones is phosphorylated by protein kinase A (PKA), as demonstrated by in vitro protein kinase assay and Western blotting with a polyclonal antibody specific against phosphorylated Ser229. Glutamate, high concentrations of K+ or K+ channel blockers known to increase neurotransmission all stimulated the phosphorylation of Kv1.4 at Ser229 via N-methyl-D-aspartate (NMDA), but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor, whereas tetradotoxin (TTX), known to block neuronal transmission, and depletion of extracellular Ca2+ inhibited phosphorylation induced by tetraethylammonium (TEA), a non-selective K+ channel blocker. Mutation of Ser229 to Ala229 enhanced the current density. Taken together, elevation of the neuronal transmission stimulates the phosphorylation of Kv1.4 at Ser229 via the Ca2+ influx through NMDA receptor. Thus, it is possible that neuronal transmission regulates neuronal excitability partially through the phosphorylation of Kv1.4S229.     

Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five- fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage- gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.     

Subcellular segregation of two A-type K+ channel proteins in rat central neurons.

In the mammalian nervous system, K+ channels regulate diverse aspects of neuronal function and are encoded by a large set of K+ channel genes. The roles of different K+ channel proteins could be dictated by their localization to specific subcellular domains. We report that two K+ channel polypeptides, Kv1.4 and Kv4.2, which form transient (A-type) K+ channels when expressed in Xenopus oocytes, are segregated in rat central neurons. Kv1.4 protein is targeted to axons and possibly terminals, while Kv4.2 is concentrated in dendrites and somata. This differential distribution implies distinct roles for these channel proteins in vivo. Their localizations suggest that Kv1.4 and Kv4.2 may regulate synaptic transmission via presynaptic, or postsynaptic mechanisms, respectively.     

Altered K(+) channel gene expression in diabetic rat ventricle: isoform switching between Kv4.2 and Kv1.4

Expression of voltage-gated K(+) channels encoding the K(+) independent transient outward current in the streptozocin-induced diabetic (DM) rat ventricle was studied to determine the basis for slowed cardiac repolarization in diabetes mellitus. Although hypertrophy was not detected in diabetic rats at 12 wk after streptozocin treatment, ventricular Kv4.2 mRNA levels decreased 41% relative to nondiabetic controls. Kv1.4 mRNA levels increased 179% relative to controls, whereas Kv4.3 mRNA levels were unaffected. Immunohistochemistry and Western blot analysis of the diabetic heart showed that the density of the Kv4.2 protein decreased, whereas Kv1.4 protein increased. Thus isoform switching from Kv4.2 to Kv1.4 is most likely the mechanism underlying the slower kinetics of transient outward K(+) current observed in the diabetic ventricle. Brain Kv1.4, Kv4.2, or Kv4.3 mRNA levels were unaffected by diabetes. Myosin heavy chain (MHC) gene expression was altered with a 32% decrease in alpha-MHC mRNA and a 259% increase in beta-MHC mRNA levels in diabetic ventricle. Low-dose insulin-like growth factor-II (IGF-II) treatment during the last 6 of the 12 wk of diabetes (DM + IGF) protected against these changes in MHC mRNAs despite continued hyperglycemia and body weight loss. IGF-II treatment did not change K(+) channel mRNA levels in DM or control rat ventricles. Thus IGF treatment may prevent some, but not all, biochemical abnormalities in the diabetic heart.     

Disinactivation of N-type inactivation of voltage-gated K channels by an erbstatin analogue

In some A-type voltage-gated K channels, rapid inactivation is achieved through the binding of an N-terminal domain of the pore-forming alpha-subunit or an associated beta-subunit to a cytoplasmic acceptor located at or near the channel pore using the ball-and-chain machinery (1-5). This inactivation involving the N terminus is known as N-type inactivation. Here, we describe an erbstatin (Erb) analogue as a small molecule inhibitor of the N-type inactivation in channels of Kv1.4 and Kv1.1+Kvbeta1. We show that this inhibition of inactivation (designated as "disinactivation") is potent and selective for N-type inactivation in heterologous cells (Chinese hamster ovary and Xenopus oocytes) expressing these A-type channels. In Chinese hamster ovary cells, Erb increased the inactivation time constant of Kv1.4 from 86.5 +/- 9.5 to 150 +/- 10 ms (n = 6, p < 0.0 1). Similarly, Erb increased the inactivation time constant of Kv1.1+Kvbeta1 from 10 +/- 0.9 to 49.3 +/- 7 ms (n = 7, p < 0.01). The EC(50) for disinactivating Kv1.1+Kvbeta1 was 10.4 +/- 0.9 microm (n = 2-9). Erb had no effect upon another A-channel, Kv4.3, which does not utilize the ball-and-chain mechanism. The mechanism of Erb-induced disinactivation was also investigated. Neither cysteine oxidation nor tyrosine kinase inhibition was involved. The results demonstrate that Erb can be used as a base structure to identify potent, selective small molecule inhibitors of intracellular protein-protein interactions, and that these disinactivators may offer another therapeutic approach to the treatment of seizure disorders.     

Truncated K+ channel DNA sequences specifically suppress lymphocyte K+ channel gene expression.

We have constructed a series of deletion mutants of Kv1.3, a Shaker-like, voltage-gated K+ channel, and examined the ability of these truncated mutants to form channels and to specifically suppress full-length Kv1.3 currents. These constructs were expressed heterologously in both Xenopus oocytes and a mouse cytotoxic T cell line. Our results show that a truncated mutant Kv1.3 must contain both the amino terminus and the first transmembrane-spanning segment, S1, to suppress full-length Kv1.3 currents. Amino-terminal-truncated DNA sequences from one subfamily suppress K+ channel expression of members of only the same subfamily. The first 141 amino acids of the amino-terminal of Kv1.3 are not necessary for channel formation. Deletion of these amino acids yields a current identical to that of full-length Kv1.3, except that it cannot be suppressed by a truncated Kv1.3 containing the amino terminus and S1. To test the ability of truncated Kv1.3 to suppress endogenous K+ currents, we constructed a plasmid that contained both truncated Kv1.3 and a selection marker gene (mouse CD4). Although constitutively expressed K+ currents in Jurkat (a human T cell leukemia line) and GH3 (an anterior pituitary cell line) cells cannot be suppressed by this double-gene plasmid, stimulated (up-regulated) Shaker-like K+ currents in GH3 cells can be suppressed.     

Dexamethasone increases potassium channel messenger RNA and activity in clonal pituitary cells.

Glucocorticoid hormones are released as part of the stress response and regulate secretion by the pituitary. Since the activity of ion channels also influences secretion, we examined the effect of the glucocorticoid agonist dexamethasone on ion channel expression. K+ channel mRNA was detected in rat hypothalamus and anterior pituitary, with probes derived from the rat Kv1 gene, a member of the mammalian voltage-gated K+ channel superfamily. High levels were also detected in PRL-secreting clonal (GH3 and GH4C1) rat pituitary cells. Dexamethasone rapidly increased the steady state concentration of Kv1 mRNA in GH3 cells in a dose-dependent manner. This change in gene expression was accompanied by an increase in whole cell voltage-gated K+ current [lk(i)] with similar pharmacology to the Kv1 gene product. Our findings indicate that hormones may act directly on excitable cells to produce long term effects on electrical activity and secretion by regulating K+ channel expression.     

The sigma receptor as a ligand-regulated auxiliary potassium channel subunit

The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.     

Heteromeric Kv1 potassium channel expression: amino acid determinants involved in processing and trafficking to the cell surface

Kv1.4 and Kv1.1 potassium channels are expressed in brain as mature glycoproteins that are trans-Golgi glycosylated. When expressed in cell lines these homomers had very different trans-Golgi glycosylation efficiencies and cell surface expression levels with Kv1.4 > Kv1.1 for both parameters (Zhu, J., Watanabe, I., Gomez, B., and Thornhill, W. B. (2001) J. Biol. Chem. 276, 39419-39427). This previous study identified determinants in the outer pore region of Kv1.4 and Kv1.1 that positively and negatively, respectively, affected these events when expressed as homomers. Here we investigated which subunit exhibited positive or negative effects on these processes when expressed as heteromers. Kv1.4/Kv1.1 heteromers, by coexpression or expression as tandem-linked heteromers, were expressed on the cell surface at approximately 20-fold lower levels versus Kv1.4 homomers but they were trans-Golgi glycosylated. The lower Kv1.4/Kv1.1 expression level was not rescued by Kvbeta 2.1 subunits. Thus Kv1.1 inhibited high cell surface expression and partially retained the heteromer in the endoplasmic reticulum, whereas Kv1.4 stimulated trans-Golgi glycosylation. The subunit determinants and cellular events responsible for these differences were investigated. In a Kv1.4/Kv1.1 heteromer, the Kv1.1 pore was a major negative determinant, and it inhibited high cell surface expression because it induced high partial endoplasmic reticulum retention and it decreased protein stability. Other Kv1.1 regions also inhibited high surface expression of heteromers. The Kv1.1 C terminus induced partial Golgi retention and contributed to a decreased protein stability, whereas the Kv1.1 N terminus contributed to only a decreased protein stability. Thus a neuron may regulate its cell surface K+ channel protein levels by different Kv1 subfamily homomeric and heteromeric combinations that affect intracellular retention characteristics and protein stability.     

Solid support membranes for ion channel arrays and sensors: application to rapid screening of pharmacological compounds

The use of solid supported membranes (SSM) was investigated for reconstitution of ion channels and for potential application to screen pharmacological reagents affecting ion channel function. The voltage-gated Kv1.5 K+ channel was reconstituted on an SSM and a current was measured. This current was dependent on the presence of K+, but not Na+, indicating that the Kv1.5 K+ channel maintained cation specificity when reconstituted on SSM. Two pharmacological reagents applied to Kv1.5 K+ channels reconstituted on SSM had similar inhibitory effects as those measured using Kv1.5 in biological membranes. SSM-mounted ion channels were stable enough to be washed with buffer solution and reused many times, allowing solution exchange essential for pharmacological drug screening.     

Functional expression of a GFP-tagged Kv1.5 alpha-subunit in mouse ventricle

The experiments here were undertaken to determine the feasibility of increasing the cell surface expression of voltage-gated ion channels in cardiac cells in vivo and to explore the functional consequences of ectopic channel expression. Transgenic mice expressing a green fluorescent protein (GFP)-tagged, voltage-gated K+ (Kv) channel alpha-subunit, Kv1.5-GFP, driven by the cardiac-specific alpha-MHC promoter, were generated. In recent studies, Kv1.5 has been shown to encode the micromolar 4-aminopyridine (4-AP)-sensitive delayed rectifier K+ current (I(K,slow)) in mouse myocardium. Unexpectedly, Kv1.5-GFP expression is heterogeneous in the ventricles of these animals. Although no electrocardiographic abnormalities were evident, expression of Kv1.5-GFP results in marked decreases in action potential durations in GFP-positive ventricular myocytes. In voltage-clamp recordings from GFP-positive ventricular myocytes, peak outward K+ currents are significantly higher, and their waveforms are distinct from those recorded from wild-type cells. Pharmacological experiments revealed a selective increase in a micromolar 4-AP-sensitive current, similar to the 4-AP-sensitive component of I(K,slow) in wild-type cells. The inactivation rate of the "overexpressed" current, however, is significantly slower than the Kv1.5-encoded component of I(K,slow) in wild-type cells, suggesting differences in association with accessory subunits and/or posttranslational processing.     

Molecular cloning and functional characterization of a novel delayed rectifier potassium channel from channel catfish (Ictalurus punctatus): expression in taste buds

The gustatory system of channel catfish is widely studied for its sensitivity to amino acids. As a first step in identifying the molecular components that play a role in taste transduction in catfish, we cloned the full-length cDNA for Kv2-catfish, a novel K(+) channel that is expressed in taste buds. The deduced amino acid sequence is 816 residues, and shares a 56-59% sequence identity with Kv2.1 and Kv2.2, the other members of the vertebrate Kv2 subfamily of voltage-gated K(+) channels. The Kv2-catfish RNA was expressed in taste buds, brain, skeletal muscle, kidney, intestine and gills, and its gene is represented as a single copy in the catfish genome. Recombinant channels expressed in XENOPUS: oocytes were selective for K(+), and were inhibited by tetraethylammonium applied to the extracellular side of the membrane during two-electrode voltage clamp analysis with a 50% inhibitory constant of 6.1 mM. The channels showed voltage-dependent activation, and did not inactivate within 200 ms. Functionally, Kv2-catfish is a voltage-gated, delayed rectifier K(+) channel, and its primary structure is the most divergent sequence identified among the vertebrate members of the Kv2 subfamily of K(+) channels, being related equally well to Kv2.1 and Kv2.2.     

The P-region and S6 of Kv3.1 contribute to the formation of the ion conduction pathway.

The loop between transmembrane regions S5 and S6 (P-region) of voltage-gated K+ channels has been proposed to form the ion-conducting pore, and the internal part of this segment is reported to be responsible for ion permeation and internal tetraethylammonium (TEA) binding. The two T-cell K+ channels, Kv3.1 and Kv1.3, with widely divergent pore properties, differ by a single residue in this internal P-region, leucine 401 in Kv3.1 corresponding to valine 398 in Kv1.3. The L401V mutation in Kv3.1 was created with the anticipation that the mutant channel would exhibit Kv1.3-like deep-pore properties. Surprisingly, this mutation did not alter single channel conductance and only moderately enhanced internal TEA sensitivity, indicating that residues outside the P-region influence these properties. Our search for additional residues was guided by the model of Durell and Guy, which predicted that the C-terminal end of S6 formed part of the K+ conduction pathway. In this segment, the two channels diverge at only one position, Kv3.1 containing M430 in place of leucine in Kv1.3. The M430L mutant of Kv3.1 exhibited permeant ion- and voltage-dependent flickery outward single channel currents, with no obvious changes in other pore properties. Modification of one or more ion-binding sites located in the electric field and possibly within the channel pore could give rise to this type of channel flicker.     

Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells

Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to K+ efflux; blockade of plasmalemmal K+ channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated K+ (Kv) channels. A human bcl-2 gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents (I(Kv)). In contrast, the apoptosis inducer staurosporine (ST) enhanced I(Kv). In bcl-2-infected cells, however, the ST-induced increase in I(Kv) was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (-bcl-2). Blockade of Kv channels in control cells (-bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in I(Kv) and apoptosis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of I(Kv) and downregulated the mRNA expression of the pore-forming Kv channel alpha-subunits (Kv1.1, Kv1.5, and Kv2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.