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PubMed1997

Identification and functional characterization of a K+ channel alpha-subunit with regulatory properties specific to brain.

The physiological diversity of K+ channels mainly depends on the expression of several genes encoding different alpha-subunits. We have cloned a new K+ channel alpha-subunit (Kv2.3r) that is unable to form functional channels on its own but that has a major regulatory function. Kv2.3r can coassemble selectively with other alpha-subunits to form functional heteromultimeric K+ channels with kinetic properties that differ from those of the parent channels. Kv2.3r is expressed exclusively in the brain, being concentrated particularly in neocortical neurons. The functional expression of this regulatory alpha-subunit represents a novel mechanism without precedents in voltage-gated channels, which might contribute to further increase the functional diversity of K+ channels necessary to specify the intrinsic electrical properties of individual neurons.

Amino Acid Sequence↗
PubMed1997

Atomic distance estimates from disulfides and high-affinity metal-binding sites in a K+ channel pore.

The pore of potassium channels is lined by four identical, highly conserved hairpin loops, symmetrically arranged around a central permeation pathway. Introduction of cysteines into the external mouth of the drk1 K channel pore resulted in the formation of disulfide bonds that were incompatible with channel function. Breaking these bonds restored function and resulted in a high-affinity Cd(2+)-binding site, indicating coordinated ligation by multiple sulfhydryls. Dimeric constructs showed that these disulfide bonds formed between subunits. These results impose narrow constraints on intersubunit atomic distances in the pore that strongly support a radial pore model. The data also suggest an important functional role for the outer mouth of the pore in gating or permeation.

Amino Acid Sequence↗
PubMed1999

Identification, cloning and expression of rabbit vascular smooth muscle Kv1.5 and comparison with native delayed rectifier K+ current.

1. The molecular basis of voltage-gated, delayed rectifier K+ (KDR) channels in vascular smooth muscle cells is poorly defined. In this study we employed (i) an antibody against Kv1.5 and (ii) a cDNA clone encoding Kv1.5 derived from rabbit portal vein (RPV) to demonstrate Kv1.5 expression in RPV and to compare the properties of RPVKv1.5 expressed in mammalian cells with those of native RPV KDR current. 2. Expression of Kv1.5 channel protein in RPV was demonstrated by (i) immunocytolocalization of an antibody raised against a C-terminal epitope of mouse cardiac Kv1.5 in permeabilized, freshly isolated RPV smooth muscle cells and (ii) isolation of a cDNA clone encoding RPVKv1.5 by reverse transcription-polymerase chain reaction (RT-PCR) using mRNA derived from endothelium-denuded and adventitia-free RPV. 3. RPVKv1.5 cDNA was expressed in mammalian L cells and human embryonic kidney (HEK293) cells and the properties of the expressed channels compared with those of native KDR channels of freshly dispersed myocytes under identical conditions. 4. The kinetics and voltage dependence of activation of L cell-expressed RPVKv1.5 and native KDR current were identical, as were the kinetics of recovery from inactivation and single channel conductance. In contrast, there was little similarity between HEK293 cell-expressed RPVKv1.5 and native KDR current. 5. Inactivation occurred with the same voltage for half-maximal availability, but the kinetics and slope constant for the voltage dependence of inactivation for L cell-expressed RPVKv1.5 and the native current were different: slow time constants were 6.5 +/- 0.6 and 3.5 +/- 0.4 s and slope factors were 4.7 +/- 0.2 and 7.0 +/- 0.8 mV, respectively. 6. This study provides immunofluorescence and functional evidence that Kv1.5 alpha-subunits are a component of native KDR channels of vascular smooth muscle cells of RPV. However, the differences in kinetics and voltage sensitivity of inactivation between L cell- and HEK293 cell-expressed channels and native KDR channels provide functional evidence that vascular KDR current is not due to homomultimers of RPV Kv1.5 alone. The channel structure may be more complex, involving heteromultimers and modulatory Kvbeta-subunits, and/or native KDR current may have other components involving Kvalpha-subunits of other families.

Amino Acid Sequence↗
PubMed2005

Calcium- and metabolic state-dependent modulation of the voltage-dependent Kv2.1 channel regulates neuronal excitability in response to ischemia.

Ischemic stroke is often accompanied by neuronal hyperexcitability (i.e., seizures), which aggravates brain damage. Therefore, suppressing stroke-induced hyperexcitability and associated excitoxicity is a major focus of treatment for ischemic insults. Both ATP-dependent and Ca2+-activated K+ channels have been implicated in protective mechanisms to suppress ischemia-induced hyperexcitability. Here we provide evidence that the localization and function of Kv2.1, the major somatodendritic delayed rectifier voltage-dependent K+ channel in central neurons, is regulated by hypoxia/ischemia-induced changes in metabolic state and intracellular Ca2+ levels. Hypoxia/ischemia in rat brain induced a dramatic dephosphorylation of Kv2.1 and the translocation of surface Kv2.1 from clusters to a uniform localization. In cultured rat hippocampal neurons, chemical ischemia (CI) elicited a similar dephosphorylation and translocation of Kv2.1. These events were reversible and were mediated by Ca2+ release from intracellular stores and calcineurin-mediated Kv2.1 dephosphorylation. CI also induced a hyperpolarizing shift in the voltage-dependent activation of neuronal delayed rectifier currents (IK), leading to enhanced IK and suppressed neuronal excitability. The IK blocker tetraethylammonium reversed the ischemia-induced suppression of excitability and aggravated ischemic neuronal damage. Our results show that Kv2.1 can act as a novel Ca2+- and metabolic state-sensitive K+ channel and suggest that dynamic modulation of IK/Kv2.1 in response to hypoxia/ischemia suppresses neuronal excitability and could confer neuroprotection in response to brief ischemic insults.

Anesthesia↗
PubMed1999

Heteromeric assembly of Kv2.1 with Kv9.3: effect on the state dependence of inactivation.

Modulatory alpha-subunits of Kv channels remain electrically silent after homomeric expression. Their interactions with Kv2 alpha-subunits via the amino-terminal domain promote the assembly of heteromeric functional channels. The kinetic features of these heteromers differ from those of Kv2 homomers, suggesting a distinct role in electrical signaling. This study investigates biophysical properties of channels emerging from the coexpression of Kv2.1 with the modulatory alpha-subunit Kv9.3. Changes relative to homomeric Kv2.1 concern activation, deactivation, inactivation, and recovery from inactivation. A detailed description of Kv2.1/Kv9.3 inactivation is presented. Kv2.1/Kv9.3 heteromers inactivate in a fast and complete fashion from intermediate closed states, but in a slow and incomplete manner from open states. Intermediate closed states of channel gating can be approached through partial activation or deactivation, according to a proposed qualitative model. These transitions are rate-limiting for Kv2.1/Kv9.3 inactivation. Finally, based on the kinetic description, we propose a putative function for Kv2.1/Kv9.3 heteromers in rat heart.

Animals↗
PubMed1999

Expression of voltage-dependent K(+) channel genes in mesenteric artery smooth muscle cells.

Molecular basis of native voltage-dependent K(+) (Kv) channels in smooth muscle cells (SMCs) from rat mesenteric arteries was investigated. The whole cell patch-clamp study revealed that a 4-aminopyridine-sensitive delayed rectifier K(+) current (I(K)) was the predominant K(+) conductance in these cells. A systematic screening of the expression of 18 Kv channel genes using RT-PCR technique showed that six I(K)-encoding genes (Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv2.2, and Kv3.2) were expressed in mesenteric artery. Although no transient outward Kv currents (I(A)) were recorded in the studied SMCs, transcripts of multiple I(A)-encoding genes, including Kv1.4, Kv3.3, Kv3.4, Kv4.1, Kv4.2, and Kv4.3 as well as I(A)-facilitating Kv beta-subunits (Kvbeta1, Kvbeta2, and Kvbeta3), were detected in mesenteric arteries. Western blot analysis demonstrated that four I(K)-related Kv channel proteins (Kv1.2, Kv1. 3, Kv1.5, and Kv2.1) were detected in mesenteric artery tissues. The presence of Kv1.2, Kv1.3, Kv1.5, and Kv2.1 channel proteins in isolated SMCs was further confirmed by immunocytochemistry study. Our results suggest that the native I(K) in rat mesenteric artery SMCs might be generated by heteromultimerization of Kv genes.

4-Aminopyridine↗
PubMed2005

Open form of syntaxin-1A is a more potent inhibitor than wild-type syntaxin-1A of Kv2.1 channels.

We have shown that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins not only participate directly in exocytosis, but also regulate the dominant membrane-repolarizing Kv channels (voltage-gated K+ channels), such as Kv2.1, in pancreatic beta-cells. In a recent report, we demonstrated that WT (wild-type) Syn-1A (syntaxin-1A) inhibits Kv2.1 channel trafficking and gating through binding to the cytoplasmic C-terminus of Kv2.1. During beta-cell exocytosis, Syn-1A converts from a closed form into an open form which reveals its active H3 domain to bind its SNARE partners SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin. In the present study, we compared the effects of the WT Syn-1A and a mutant open form Syn-1A (L165A, E166A) on Kv2.1 channel trafficking and gating. When co-expressed in HEK-293 cells (human embryonic kidney-293 cells), the open form Syn-1A decreased Kv2.1 current density more than (P<0.05) the WT Syn-1A (166+/-35 and 371+/-93 pA/pF respectively; control=911+/-91 pA/pF). Confocal microscopy and biotinylation experiments showed that both the WT and open form Syn-1A inhibited Kv2.1 expression at the plasma membrane to a similar extent, suggesting that the stronger reduction of Kv2.1 current density by the open form compared with the WT Syn-1A is probably due to a stronger direct inhibition of channel activity. Consistently, dialysis of the recombinant open form Syn-1A protein into Kv2.1-expressing HEK-293 cells caused stronger inhibition of Kv2.1 current amplitude (P<0.05) than the WT Syn-1A protein (73+/-2 and 82+/-3% of the control respectively). We found that the H3 but not H(ABC) domain is the putative active domain of Syn-1A, which bound to and inhibited the Kv2.1 channel. When co-expressed in HEK-293 cells, the open-form Syn-1A slowed down Kv2.1 channel activation (tau=12.3+/-0.8 ms) much more than (P<0.05) WT Syn-1A (tau=7.9+/-0.8 ms; control tau=5.5+/-0.6 ms). In addition, only the open form Syn-1A, but not the WT Syn-1A, caused a significant (P<0.05) left-shift in the steady-state inactivation curve (V(1/2)=33.1+/-1.3 and -29.4+/-1.1 mV respectively; control V(1/2)=-24.8+/-2 mV). The present study therefore indicates that the open form of Syn-1A is more potent than the WT Syn-1A in inhibiting the Kv2.1 channel. Such stronger inhibition by the open form of Syn-1A may limit K+ efflux and thus decelerate membrane repolarization during exocytosis, leading to optimization of insulin release.

Antigens, Surface↗
PubMed2001

Astrocyte-released cytokines induce ramification and outward K+ channel expression in microglia via distinct signalling pathways.

Differentiation of microglial cells is characterized by transformation from ameboid into ramified cell shape and up-regulation of K+ channels. The processes of microglial differentiation are controlled by astrocytic factors. The mechanisms by which astrocytes cause developmental changes in morphological and electrophysiological properties of microglia have remained unclear. We show here that the cytokines transforming growth factor-beta (TGF-beta), macrophage colony-stimulating factor (M-CSF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) are released by astrocytes at concentrations sufficient to induce ramification and up-regulation of delayed rectifier (DR) K+ channels in microglia. Transformation from ameboid into ramified morphology induced in microglia by exposure to astrocyte-conditioned medium (ACM) was inhibited by neutralizing antibodies against TGF-beta, M-CSF or GM-CSF, whilst ACM-induced DR channel expression was exclusively inhibited by antibodies against TGF-beta. Although both ramification and DR channel up-regulation occurred simultaneously, DR channel blockade by charybdotoxin failed to inhibit microglial ramification. The ACM-induced ramification of microglia was inhibited by the tyrosine kinase inhibitor genistein, whereas DR channel up-regulation did not occur in the presence of the serine/threonine kinase inhibitor H7. Our data suggest that astrocytes modulate processes of microglial differentiation in parallel but via distinct signalling pathways.

Animals↗
PubMed1999

Kv3.1-Kv3.2 channels underlie a high-voltage-activating component of the delayed rectifier K+ current in projecting neurons from the globus pallidus.

The globus pallidus plays central roles in the basal ganglia circuitry involved in movement control as well as in cognitive and emotional functions. There is therefore great interest in the anatomic and electrophysiological characterization of this nucleus. Most pallidal neurons are GABAergic projecting cells, a large fraction of which express the calcium binding protein parvalbumin (PV). Here we show that PV-containing pallidal neurons coexpress Kv3. 1 and Kv3.2 K+ channel proteins and that both Kv3.1 and Kv3.2 antibodies coprecipitate both channel proteins from pallidal membrane extracts solubilized with nondenaturing detergents, suggesting that the two channel subunits are forming heteromeric channels. Kv3.1 and Kv3.2 channels have several unusual electrophysiological properties when expressed in heterologous expression systems and are thought to play special roles in neuronal excitability including facilitating sustained high-frequency firing in fast-spiking neurons such as interneurons in the cortex and the hippocampus. Electrophysiological analysis of freshly dissociated pallidal neurons demonstrates that these cells have a current that is nearly identical to the currents expressed by Kv3.1 and Kv3.2 proteins in heterologous expression systems, including activation at very depolarized membrane potentials (more positive than -10 mV) and very fast deactivation rates. These results suggest that the electrophysiological properties of native channels containing Kv3.1 and Kv3.2 proteins in pallidal neurons are not significantly affected by factors such as associated subunits or postranslational modifications that result in channels having different properties in heterologous expression systems and native neurons. Most neurons in the globus pallidus have been reported to fire sustained trains of action potentials at high-frequency. Kv3.1-Kv3.2 voltage-gated K+ channels may play a role in helping maintain sustained high-frequency repetitive firing as they probably do in other neurons.

Animals↗
PubMed1999

Mutational analysis of the Shab-encoded delayed rectifier K(+) channels in Drosophila.

K(+) currents in Drosophila muscles have been resolved into two voltage-activated currents (I(A) and I(K)) and two Ca(2+)-activated currents (I(CF) and I(CS)). Mutations that affect I(A) (Shaker) and I(CF) (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (I(K)) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K(+) channel gene, we show that this gene encodes the delayed rectifier K(+) channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K(+) current (I(K)). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivo physiological role for the Shab-encoded K(+) channels in Drosophila. The availability of mutations that affect I(K) opens up possibilities for studying I(K) and its role in larval muscle excitability.

Amino Acid Sequence↗
PubMed2005

Inhibitory effect of thiopental on ultra-rapid delayed rectifier K+ current in H9c2 cells.

Using the whole-cell voltage clamp technique, we investigated the effects of thiopental on membrane currents in H9c2 cells, a cell line derived from embryonic rat heart. Thiopental blocked a rapidly activating, very slowly-inactivating ultra-rapid type I(Kur)-like outward K(+) current in a concentration-dependent manner. The half-maximal concentration (IC(50)) of thiopental was 97 microM with a Hill coefficient of 1.2. The thiopental-sensitive current was also blocked by high concentrations of nifedipine (IC(50) = 9.1 microM) and 100 microM chromanol 293B, a blocker of slowly activating delayed rectifier K+ current (I(Ks)), but was insensitive to E-4031, an inhibitor of rapidly activating delayed rectifier K(+) current (I(Kr)). TEA (tetraethylammonium) at 5 mM and 4-AP (4-aminopiridine) at 1 mM reduced the K(+) current to 30.8 +/- 12.2% and 20.5 +/- 6.5% of the control, respectively. Using RT-PCR, we detected mRNAs of Kv2.1, Kv3.4, Kv4.1, and Kv4.3 in H9c2 cells. Among those, Kv2.1 and Kv3.4 have I(Kur)-type kinetics and are therefore candidates for thiopental-sensitive K(+) channels in H9c2 cells. This is the first report showing that thiopental inhibits I(Kur). This effect of thiopental may be involved in its reported prolongation of cardiac action potentials.

Anesthetics↗
PubMed1998

Modulation of C-type inactivation by K+ at the potassium channel selectivity filter.

With prolonged or repetitive activation, voltage-gated K+ channels undergo a slow (C-type) inactivation mechanism, which decreases current flow through the channel. Previous observations suggest that C-type inactivation results from a localized constriction in the outer mouth of the channel pore and that the rate of inactivation is controlled by the-rate at which K+ leaves an unidentified binding site in the pore. We have functionally identified two K+ binding sites in the conduction pathway of a chimeric K+ channel that conducts Na+ in the absence of K+. One site has a high affinity for K+ and contributes to the selectivity filter mechanism for K+ over Na+. Another site, external to the high-affinity site, has a lower affinity for K+ and is not involved in channel selectivity. Binding of K+ to the high-affinity binding site slowed inactivation. Binding of cations to the external low-affinity site did not slow inactivation directly but could slow it indirectly, apparently by trapping K+ at the high-affinity site. These data support a model whereby C-type inactivation involves a constriction at the selectivity filter, and the constriction cannot proceed when the selectivity filter is occupied by K+.

Binding Sites↗
PubMed1998

Influence of non-P region domains on selectivity filter properties in voltage-gated K+ channels.

The selectivity filter in voltage-gated K+ channels is formed at the interface of the pore loops (S5-S6 loop) from four channel subunits. Whereas most K+ channels are essentially impermeable to Na+, the Kv2.1 K+ channel conducts Na+ relatively well in the absence of K+ and selects for K+ over Na+ at least partially by an affinity-based competition mechanism. To examine whether the ability of Kv2.1 to conduct Na+ reflected unique properties of either its S5-S6 loop or channel domains that held the S5-S6 loop in place (the scaffolding), we studied chimeras made from Kv1.3 (which is completely impermeable to Na+) and Kv2.1. Chimeras that contained either the S5-S6 loop from Kv1.3 inserted into the Kv2.1 scaffolding or vice versa both made highly selective K+ channels that conducted Na+ and displayed competition between Na+ and K+ for conduction through the pore: In channels that contained the S5-S6 loop from Kv2.1, concentration-dependent block of Na+ current by either external or internal K+ differed depending on whether Kv2.1 or Kv1.3 donated the scaffolding. These results indicate that neither the S5-S6 loop nor the scaffolding from Kv2.1 possess unique attributes that permit Na+ to conduct through the channel. Furthermore, these results indicate that the competitive interaction between K+ and Na+ at the selectivity filter is determined not only by the S5-S6 loop but also by the scaffolding that holds the S5-S6 loop.

Cations↗
PubMed2001

Activation of melatonin receptor increases a delayed rectifier K+ current in rat cerebellar granule cells.

The present study was initiated to investigate the effect of melatonin on K+ current in rat cerebellar granule cells for 2 to 6 days in culture (DIC). The whole-cell configuration of the conventional patch-clamp technique was used to record the outward K+ current. Two types of outward K+ current, a transient outward K+ current and a delayed rectifier K+ current, were separated by different voltage protocols and a specific blocker of K+ channel. Application of melatonin (10 microM) by a brief pressure ejection induced a significant and reversible increase of the delayed rectifier K+ current amplitude in 78% of the cells tested. The activated effect of melatonin on the K+ current was independent of the time in culture, and the percentage of activation remained at a relatively stable level from 2 DIC to 6 DIC; but that was dependent on the concentration of melatonin applied. The activation of the K+ current induced by melatonin presented no desensitization after repeated application of melatonin. The effect of melatonin on the K+ current can be mimicked by 2-iodomelatonin, a melatonin receptor agonist. With the addition of guanosine-5'-O-(3-thiophosphate) in the pipette solution, melatonin caused a stronger activation effect on the K+ channels, and an irreversible increase of the current amplitude in some granule cells tested. Pretreatment of cells with PTX suppressed the action of melatonin on the K+ current in most granule cells studied. In addition, the activation curves and inactivation curves tested with the steady-state activation and inactivation protocols were unchanged by melatonin, suggesting that melatonin did not modulate the channel's activation and inactivation properties. Our results demonstrated the presence of a functional melatonin receptor in cultured cerebellar granule cells from neonatal cerebellum. Activating the receptor can modulate the outward K+ currents by coupling to a PTX-sensitive G protein.

Animals↗
PubMed1996

Expression and function of pancreatic beta-cell delayed rectifier K+ channels. Role in stimulus-secretion coupling.

Voltage-dependent delayed rectifier K+ channels regulate aspects of both stimulus-secretion and excitation-contraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (betaTC3-neo) and beta-cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion. Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expression of the K+ channel gene Kv2.1 in betaTC3-neo insulinoma cells and purified rodent pancreatic beta-cells was detected and confirmed by immunoblotting in the insulinoma cells. betaTC3-neo cells were also found to express a related K+ channel, Kv3.2. Whole-cell patch clamp demonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar Kd values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents. The effect of these blockers on intracellular Ca2+ concentration ([Ca2+]i) was studied with fura-2 microspectrofluorimetry and imaging techniques. In the absence of glucose, exposure to TEA (1-20 mM) had minimal effects on betaTC3-neo or rodent islet [Ca2+]i, but in the presence of glucose, TEA activated large amplitude [Ca2+]i oscillations. In the insulinoma cells the TEA-induced [Ca2+]i oscillations were driven by synchronous oscillations in membrane potential, resulting in a 4-fold potentiation of insulin secretion. Activation of specific delayed rectifier K+ channels can therefore suppress stimulus-secretion coupling by damping oscillations in membrane potential and [Ca2+]i and thereby regulate secretion. These studies implicate previously uncharacterized beta-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+]i, and insulin secretion.

4-Aminopyridine↗
PubMed1998

A novel benzodiazepine that activates cardiac slow delayed rectifier K+ currents.

The slowly activating delayed rectifier K+ current, IKs, is an important modulator of cardiac action potential repolarization. Here, we describe a novel benzodiazepine, [L-364,373 [(3-R)-1, 3-dihydro-5-(2-fluorophenyl)-3-(1H-indol-3-ylmethyl)-1-methyl-2H- 1,4-benzodiazepin-2-one] (R-L3), that activates IKs and shortens action potentials in guinea pig cardiac myocytes. These effects were additive to isoproterenol, indicating that channel activation by R-L3 was independent of beta-adrenergic receptor stimulation. The increase of IKs by R-L3 was stereospecific; the S-enantiomer, S-L3, blocked IKs at all concentrations examined. The increase in IKs by R-L3 was greatest at voltages near the threshold for normal channel activation, caused by a shift in the voltage dependence of IKs activation. R-L3 slowed the rate of IKs deactivation and shifted the half-point of the isochronal (7.5 sec) activation curve for IKs by -16 mV at 0.1 microM and -24 mV at 1 microM. R-L3 had similar effects on cloned KvLQT1 channels expressed in Xenopus laevis oocytes but did not affect channels formed by coassembly of KvLQT1 and hminK subunits. These findings indicate that the association of minK with KvLQT1 interferes with the binding of R-L3 or prevents its action once bound to KvLQT1 subunits.

Action Potentials↗
PubMed1999

Subunit assembly and domain analysis of electrically silent K+ channel alpha-subunits of the rat Kv9 subfamily.

Alpha-subunits of the voltage-gated potassium channel (Kv) subfamily Kv9 show no channel activity after homomultimeric expression in heterologous expression systems. This report shows that heteromultimeric expression of rKv9.1 and rKv9.3 specifically suppresses the currents mediated by alpha-subunits of the Kv2 and Kv3 subfamilies but does not affect currents mediated by alpha-subunits of the Kv1 and Kv4 subfamilies. To understand the molecular basis of the electrical silence of Kv9 homomultimeric channels, crucial functional domains (amino and carboxy terminus, S4 segment, and pore region) were exchanged between Kv9 alpha-subunits and rKv1.3. Electrophysiological studies of these chimeras revealed that the pore region is involved in determining the nonconductive behavior of homomultimeric Kv9 channels. This analysis was extended by protein interaction assays, aiming to identify the region of Kv9 subunits responsible for the specific suppression of rKv2.1- and rKv3.4-mediated currents. We could show that the amino-terminal domain of Kv9 alpha-subunits does not support homomultimeric assembly but interacts specifically with the rKv2.1 amino-terminal region. Conversely, the specific intersubfamily assembly of rKv3.4 with rKv9.1 or rKv9.3 is governed by the hydrophobic core and not the amino-terminal domain.

Amino Acid Sequence↗
PubMed2002

O2-sensitive K+ channels in immortalised rat chromaffin-cell-derived MAH cells.

The regulation of K(+) channels by O(2) levels is a key link between hypoxia and neurotransmitter release in neuroendocrine cells. Here, we examined the effects of hypoxia on K(+) channels in the immortalised v-myc, adrenal-derived HNK1(+) (MAH) cell line. MAH cells possess a K(+) conductance that is sensitive to Cd(2+), iberiotoxin and apamin, and which is inhibited by ~24 % when exposed to a hypoxic perfusate (O(2) tension 20 mmHg). This conductance was attributed to high-conductance Ca(2+)-activated K(+) (BK) and small-conductance Ca(2+)-activated K(+) (SK) channels, which are major contributors to the O(2)-sensitive K(+) conductance in adrenomedullary chromaffin cells. Under low [Ca(2+)](i) conditions that prevented activation of Ca(2+)-dependent K(+) conductances, a rapidly activating and slowly inactivating K(+) conductance, sensitive to both TEA and 4-aminopyridine (4-AP), but insensitive to 100 nM charybdotoxin (CTX), was identified. This current was also reduced (by ~25 %) when exposed to hypoxia. The hypoxia-sensitive component of this current was greatly attenuated by 10 mM 4-AP, but was only slightly reduced by 10 mM TEA. This suggests the presence of delayed-rectifier O(2)-sensitive channels comprising homomultimeric Kv1.5 or heteromultimeric Kv1.5/Kv1.2 channel subunits. The presence of both Kv1.5 and Kv1.2 alpha-subunits was confirmed using immunocytochemical techniques. We also demonstrated that these K(+) channel subunits are present in neonatal rat adrenomedullary chromaffin cells in situ. These data indicate that MAH cells possess O(2)-sensitive K(+) channels with characteristics similar to those observed previously in isolated chromaffin cells, and therefore provide an excellent model for examining the cellular mechanisms of O(2) sensing in adrenomedullary chromaffin cells.

Adrenal Glands↗