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Results for “Delayed Rectifier Potassium Channels”

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PubMed2006

AKAPs as antiarrhythmic targets?

Phosphorylation of ion channels plays a critical role in the modulation and amplification of biophysical signals. Kinases and phosphatases have broad substrate recognition sequences. Therefore, the targeting of kinases and phosphatases to specific sites enhances the regulation of diverse signaling events. Ion channel macromolecular complexes can be formed by the association of A-kinase anchoring proteins (AKAPs) or other adaptor proteins directly with the channel. The discovery that leucine/isoleucine zippers play an important role in the recruitment of phosphorylation-modulatory proteins to certain ion channels has permitted the elucidation of specific ion channel macromolecular complexes. Disruption of signaling complexes by genetic defects can lead to abnormal physiological function. This chapter will focus on evidence supporting the concept that ion channel macromolecular complex formation plays an important role in regulating channel function in normal and diseased states. Moreover, we demonstrate that abnormal complex formation may directly lead to abnormal channel regulation by cellular signaling pathways, potentially leading to arrhythmogenesis and cardiac dysfunction.

A Kinase Anchor Proteins↗

PubMed2002

Gating, modulation and subunit composition of voltage-gated K(+) channels in dendritic inhibitory interneurones of rat hippocampus.

GABAergic interneurones are diverse in their morphological and functional properties. Perisomatic inhibitory cells show fast spiking during sustained current injection, whereas dendritic inhibitory cells fire action potentials with lower frequency. We examined functional and molecular properties of K(+) channels in interneurones with horizontal dendrites in stratum oriens-alveus (OA) of the hippocampal CA1 region, which mainly comprise somatostatin-positive dendritic inhibitory cells. Voltage-gated K(+) currents in nucleated patches isolated from OA interneurones consisted of three major components: a fast delayed rectifier K(+) current component that was highly sensitive to external 4-aminopyridine (4-AP) and tetraethylammonium (TEA) (half-maximal inhibitory concentrations < 0.1 mM for both blockers), a slow delayed rectifier K(+) current component that was sensitive to high concentrations of TEA, but insensitive to 4-AP, and a rapidly inactivating A-type K(+) current component that was blocked by high concentrations of 4-AP, but resistant to TEA. The relative contributions of these components to the macroscopic K(+) current were estimated as 57 +/- 5, 25 +/- 6, and 19 +/- 2 %, respectively. Dendrotoxin, a selective blocker of Kv1 channels had only minimal effects on K(+) currents in nucleated patches. Coapplication of the membrane-permeant cAMP analogue 8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate (cpt-cAMP) and the phosphodiesterase blocker isobutyl-methylxanthine (IBMX) resulted in a selective inhibition of the fast delayed rectifier K(+) current component. This inhibition was absent in the presence of the protein kinase A (PKA) inhibitor H-89, implying the involvement of PKA-mediated phosphorylation. Single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed a high abundance of Kv3.2 mRNA in OA interneurones, whereas the expression level of Kv3.1 mRNA was markedly lower. Similarly, RT-PCR analysis showed a high abundance of Kv4.3 mRNA, whereas Kv4.2 mRNA was undetectable. This suggests that the fast delayed rectifier K(+) current and the A-type K(+) current component are mediated predominantly by homomeric Kv3.2 and Kv4.3 channels. Selective modulation of Kv3.2 channels in OA interneurones by cAMP is likely to be an important factor regulating the activity of dendritic inhibitory cells in principal neurone-interneurone microcircuits.

Action Potentials↗

PubMed2002

Electrophysiological consequences of human IKs channel expression in adult murine heart.

We expressed human delayed rectifier K(+) cardiac current (I(Ks)) channels in the murine heart, which lacks native I(Ks), to determine their electrophysiological role. Mice expressing human I(Ks) channels were anesthetized, and an electrocardiogram and monophasic action potentials (MAP) recorded from the left ventricle. Sinus rate was not different between wild-type mice (WT) and transgenic mice (TG). Infusion of isoproterenol accelerated WT heart rate but not TG. Lack of TG sinus rate responsiveness may have resulted from accumulated outward current in I(Ks) channels in sinus node. Ventricular MAP duration of TG mice to 50% repolarization (APD(50)) during ventricular pacing was shorter than WT, likely resulting from outward current through I(Ks) channels. TG APD(50) showed enhanced responsiveness (shortening) to isoproterenol compared with WT. Ventricular tachyarrhythmias were initiated in TG mice by programmed stimulation but not in WT and were accelerated by isoproterenol. I(Ks) channels impart beta-adrenergic sensitivity to the ventricles and may be responsible for ventricular tachyarrhythmias.

Adrenergic beta-Agonists↗

PubMed2003

Phosphorylation-dependent regulation of Kv2.1 Channel activity at tyrosine 124 by Src and by protein-tyrosine phosphatase epsilon.

Voltage-gated potassium (Kv) channels are a complex and heterogeneous family of proteins that play major roles in brain and cardiac excitability. Although Kv channels are activated by changes in cell membrane potential, tyrosine phosphorylation of channel subunits can modulate the extent of channel activation by depolarization. We have previously shown that dephosphorylation of Kv2.1 by the nonreceptor-type tyrosine phosphatase PTPepsilon (cyt-PTPepsilon) down-regulates channel activity and counters its phosphorylation and up-regulation by Src or Fyn. In the present study, we identify tyrosine 124 within the T1 cytosolic domain of Kv2.1 as a target site for the activities of Src and cyt-PTPepsilon. Tyr(124) is phosphorylated by Src in vitro; in whole cells, Y124F Kv2.1 is significantly less phosphorylated by Src and loses most of its ability to bind the D245A substrate-trapping mutant of cyt-PTPepsilon. Phosphorylation of Tyr(124) is critical for Src-mediated up-regulation of Kv2.1 channel activity, since Y124F Kv2.1-mediated K(+) currents are only marginally up-regulated by Src, in contrast with a 3-fold up-regulation of wild-type Kv2.1 channels by the kinase. Other properties of Kv2.1, such as expression levels, subcellular localization, and voltage dependence of channel activation, are unchanged in Y124F Kv2.1, indicating that the effects of the Y124F mutation are specific. Together, these results indicate that Tyr(124) is a significant site at which the mutually antagonistic activities of Src and cyt-PTPepsilon affect Kv2.1 phosphorylation and activity.

Animals↗

PubMed1998

Heteromultimeric potassium channels formed by members of the Kv2 subfamily.

Four alpha-subunits are thought to coassemble and form a voltage-dependent potassium (Kv) channel. Kv alpha-subunits belong to one of four major subfamilies (Kv1, Kv2, Kv3, Kv4). Within a subfamily up to eight different genetic isotypes exist (e.g., Kv1.1, Kv1.2). Different isotypes within the Kv1 or Kv3 subfamily coassemble. It is not known, however, whether the only two members of the vertebrate Kv2 subfamily identified thus far, Kv2.1 and Kv2.2, heteromultimerize. This might account for the lack of detection of heteromultimeric Kv2 channels in situ despite the coexpression of Kv2.1 and Kv2.2 mRNAs within the same cell. To probe whether Kv2 isotypes can form heteromultimers, we developed a dominant-negative mutant Kv2.2 subunit to act as a molecular poison of Kv2 subunit-containing channels. The dominant-negative Kv2.2 suppresses formation of functional channels when it is coexpressed in oocytes with either wild-type Kv2.2 or Kv2.1 subunits. These results indicate that Kv2.1 and Kv2.2 subunits are capable of heteromultimerization. Thus, in native cells either Kv2.1 and Kv2.2 subunits are targeted at an early stage to different biosynthetic compartments or heteromultimerization otherwise is inhibited.

Amino Acid Sequence↗

PubMed2001

Heteromultimeric Kv1.2-Kv1.5 channels underlie 4-aminopyridine-sensitive delayed rectifier K(+) current of rabbit vascular myocytes.

The molecular identity of vascular delayed rectifier K(+) channels (K(DR)) is poorly characterized. Inhibition by 4-aminopyridine (4-AP) of K(DR) of rabbit portal vein (RPV) myocytes was studied by patch clamp and compared with that of channels composed of Kv1.5 and/or Kv1.2 subunits cloned from the RPV and expressed in mammalian cells. 4-AP block of K(DR) was pulse-frequency dependent, required channel activation, and was associated with a positive shift in voltage dependence of activation. 4-AP caused a voltage-dependent reduction in mean open time of K(DR). Relief of 4-AP block of whole cell currents during washout required channel activation and was unaffected by voltage. Homotetrameric Kv1.5 channels did not exhibit the shift in voltage dependence of activation exhibited by the native channels. In contrast, Kv1.2 channels displayed a shift in voltage dependence of activation, and this characteristic was also evident during 4-AP treatment when Kv1.2 was coexpressed with Kv1.5 or coupled to Kv1.5 in a tandem construct to produce heterotetrameric [Kv1.5/Kv1.2](2) channels. K(DR) currents were not sensitive to charybdotoxin, which blocks homotetrameric Kv1.2 channels. The findings of this study (1) indicate that vascular K(DR) are inhibited by 4-AP via an open-state block mechanism and trapping of the drug within the pore on channel closure and (2) provide novel evidence based on a comparison of functional characteristics that indicate the dominant form of vascular K(DR) channel complex in RPV involves the heteromultimeric association of Kv1.2 and Kv1.5 subunits.

4-Aminopyridine↗

PubMed2002

Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-dependent K(+) channels in neuroendocrine islet beta-cells through an interaction with the channel N terminus.

Insulin secretion is initiated by ionic events involving membrane depolarization and Ca(2+) entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K(+) channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K(+) currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K(+) currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.

Adenoviridae↗

PubMed2002

Dichloroacetate, a metabolic modulator, prevents and reverses chronic hypoxic pulmonary hypertension in rats: role of increased expression and activity of voltage-gated potassium channels.

BACKGROUND: Chronic hypoxic pulmonary hypertension (CH-PHT) is associated with suppressed expression and function of voltage-gated K(+) channels (Kv) in pulmonary artery (PA) smooth muscle cells (SMCs) and a shift in cellular redox balance toward a reduced state. We hypothesized that dichloroacetate (DCA), a metabolic modulator that can shift redox balance toward an oxidized state and increase Kv current in myocardial cells, would reverse CH-PHT. METHODS AND RESULTS: We studied 4 groups of rats: normoxic, normoxic+DCA (DCA 70 mg. kg(-1). d(-1) PO), chronically hypoxic (CH), and CH+DCA. CH and CH+DCA rats were kept in a hypoxic chamber (10% FiO(2)) for 2 to 3 weeks. DCA was given either at day 1 to prevent or at day 10 to reverse CH-PHT. We used micromanometer-tipped catheters and measured hemodynamics in closed-chest rats on days 14 to 18. CH+DCA rats had significantly reduced pulmonary vascular resistance, right ventricular hypertrophy, and PA remodeling compared with the CH rats. CH inhibited I(K), eliminated the acute hypoxia-sensitive I(K), and decreased Kv2.1 channel expression. In the short term, low-dose DCA (1 micromol/L) increased I(K) in CH-PASMCs. In a mammalian expression system, DCA activated Kv2.1 by a tyrosine kinase-dependent mechanism. When given long-term, DCA partially restored I(K) and Kv2.1 expression in PASMCs without altering right ventricular pyruvate dehydrogenase activity, suggesting that the beneficial effects of DCA occur by nonmetabolic mechanisms. CONCLUSIONS: DCA both prevents and reverses CH-PHT by a mechanism involving restoration of expression and function of Kv channels. DCA has previously been used in humans and may potentially be a therapeutic agent for pulmonary hypertension.

Animals↗

PubMed2006

Electrophysiological characterization of 14-benzoyltalatisamine, a selective blocker of the delayed rectifier K+ channel found in virtual screening.

14-Benzoyltalatisamine is a potent and selective blocker of the delayed rectifier K+ channel found in a computational virtual screening study. The compound was found to block the K+ channel from the extracellular side. However, it is unclear whether 14-benzoyltalatisamine shares the same block mechanism with tetraethylammonium (TEA). In order to elucidate how the hit compound found by the virtual screening interacts with the outer vestibule of the K+ channel, the effects of 14-benzoyltalatisamine and TEA on the delayed rectifier K+ current of rat dissociated hippocampal neurons were compared using whole-cell voltage-clamp recording. External application of 14-benzoyltalatisamine and TEA reversibly inhibited the current with IC50 values of 10.1+/-2.2 microM and 1.05+/-0.21 mM, respectively. 14-Benzoyltalatisamine exerted voltage-dependent inhibition, markedly accelerated the decay of the current, and caused a significant hyperpolarizing shift of the steady-state activation curve, whereas TEA caused voltage-independent inhibition, without affecting the kinetic parameters of the current. The blockade by 14-benzoyltalatisamine, but not by TEA, was significantly diminished in a high K+ (60 mM) external solution. The potency of 14-benzoyltalatisamine was markedly reduced in the presence of 15 mM TEA. The results suggest that 14-benzoyltalatisamine bind to the external pore entry of the delayed rectifier K+ channel with partial insertion into the selectivity filter, which is in conformity with that predicted by the molecular docking model in the virtual screening.

Aconitine↗

PubMed2001

Expression and function of native potassium channel [K(V)alpha1] subunits in terminal arterioles of rabbit.

1. In this study we investigated the expression and function of the K(V)alpha1 subfamily of voltage-gated K(+) channels in terminal arterioles from rabbit cerebral circulation. 2. K(+) current was measured from smooth muscle cells within intact freshly isolated arteriolar fragments. Current activated on depolarisation positive of about -45 mV and a large fraction of this current was blocked by 3,4-diaminopyridine (3,4-DAP) or 4-aminopyridine (4-AP), inhibitors of K(V) channels. Expression of cRNA encoding K(V)1.6 in Xenopus oocytes also generated a 4-AP-sensitive K(+) current with a threshold for activation near -45 mV. 3. Immunofluorescence labelling revealed K(V)1.2 to be specifically localised to endothelial cells, and K(V)1.5 and K(V)1.6 to plasma membranes of smooth muscle cells. 4. K(V) channel current in arteriolar fragments was blocked by correolide (which is specific for the K(V)alpha1 family of K(V) channels) but was resistant to recombinant agitoxin-2 (rAgTX2; which inhibits K(V)1.6 but not K(V)1.5). Heterologously expressed K(V)2.1 was resistant to correolide, and K(V)1.6 was blocked by rAgTX2. 5. Arterioles that were mildly preconstricted and depolarised by 0.1-0.3 nM endothelin-1 constricted further in response to 3,4-DAP, 4-AP or correolide, but not to rAgTX2. 6. We suggest that K(V)alpha1 channels are expressed in smooth muscle cells of terminal arterioles, underlie a major part of the voltage-dependent K(+) current, and have a physiological function to oppose vasoconstriction. K(V)alpha1 complexes without K(V)1.5 appear to be uncommon.

4-Aminopyridine↗

PubMed1999

Contribution of the selectivity filter to inactivation in potassium channels.

Voltage-gated K+ channels exhibit a slow inactivation process, which becomes an important influence on the rate of action potential repolarization during prolonged or repetitive depolarization. During slow inactivation, the outer mouth of the permeation pathway undergoes a conformational change. We report here that during the slow inactivation process, the channel progresses through at least three permeation states; from the initial open state that is highly selective for K+, the channel enters a state that is less permeable to K+ and more permeable to Na+, and then proceeds to a state that is non-conducting. Similar results were obtained in three different voltage-gated K+ channels: Kv2.1, a channel derived from Shaker (Shaker Delta A463C), and a chimeric channel derived from Kv2.1 and Kv1.3 that displays classical C-type inactivation. The change in selectivity displayed both voltage- and time-dependent properties of slow inactivation and was observed with K+ on either side of the channel. Elevation of internal [K+] inhibited Na+ conduction through the inactivating channel in a concentration-dependent manner. These results indicate that the change in selectivity filter function is an integral part of the slow inactivation mechanism, and argue against the hypothesis that the inactivation gate is independent from the selectivity filter. Thus, these data suggest that the selectivity filter is itself the inactivation gate.

Biophysical Phenomena↗

PubMed2004

Visceral periadventitial adipose tissue regulates arterial tone of mesenteric arteries.

Periadventitial adipose tissue produces vasoactive substances that influence vascular contraction. Earlier studies addressed this issue in aorta, a vessel that does not contribute to peripheral vascular resistance. We tested the hypothesis that periadventitial adipose tissue modulates contraction of smaller arteries more relevant to blood pressure regulation. We studied mesenteric artery rings surrounded by periadventitial adipose tissue from adult male Sprague-Dawley rats. The contractile response to serotonin, phenylephrine, and endothelin I was markedly reduced in intact vessels compared with vessels without periadventitial fat. The contractile response to U46619 or depolarizing high K+-containing solutions (60 mmol/L) was similar in vessels with and without periadventitial fat. The K+ channel opener cromakalim induced relaxation of vessels precontracted by serotonin but not by U46619 or high K+-containing solutions (60 mmol/L), suggesting that K+ channels are involved. The intracellular membrane potential of smooth muscle cells was more hyperpolarized in intact vessels than in vessels without periadventitial fat. Both the anticontractile effect and membrane hyperpolarization of periadventitial fat were abolished by inhibition of delayed-rectifier K+ (K(v)) channels with 4-aminopyridine (2 mmol/L) or 3,4-diaminopyridine (1 mmol/L). Blocking other K+ channels with glibenclamide (3 micromol/L), apamin (1 micromol/L), iberiotoxin (100 nmol/L), tetraethylammonium ions (1 mmol/L), tetrapentylammonium ions (10 micromol/L), or Ba2+ (3 micromol/L) had no effect. Longitudinal removal of half the perivascular tissue reduced the anticontractile effect of fat by almost 50%, whereas removal of the endothelium had no effect. We suggest that visceral periadventitial adipose tissue controls mesenteric arterial tone by inducing vasorelaxation via K(v) channel activation in vascular smooth muscle cells.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2004

PACAP inhibits delayed rectifier potassium current via a cAMP/PKA transduction pathway: evidence for the involvement of I k in the anti-apoptotic action of PACAP.

Abstract Activation of potassium (K(+)) currents plays a critical role in the control of programmed cell death. Because pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to inhibit the apoptotic cascade in the cerebellar cortex during development, we have investigated the effect of PACAP on K(+) currents in cultured cerebellar granule cells using the patch-clamp technique in the whole-cell configuration. Two types of outward K(+) currents, a transient K(+) current (I(A)) and a delayed rectifier K(+) current (I(K)) were characterized using two different voltage protocols and specific inhibitors of K(+) channels. Application of PACAP induced a reversible reduction of the I(K) amplitude, but did not affect I(A), while the PACAP-related peptide vasoactive intestinal polypeptide had no effect on either types of K(+) currents. Repeated applications of PACAP induced gradual attenuation of the electrophysiological response. In the presence of guanosine 5'-[gammathio]triphosphate (GTPgammaS), PACAP provoked a marked and irreversible I(K) depression, whereas cell dialysis with guanosine 5'-[betathio]diphosphate GDPbetaS totally abolished the effect of PACAP. Pre-treatment of the cells with pertussis toxin did not modify the effect of PACAP on I(K). In contrast, cholera toxin suppressed the PACAP-induced inhibition of I(K). Exposure of granule cells to dibutyryl cyclic adenosine monophosphate (dbcAMP) mimicked the inhibitory effect of PACAP on I(K). Addition of the specific protein kinase A inhibitor H89 in the patch pipette solution prevented the reduction of I(K) induced by both PACAP and dbcAMP. PACAP provoked a sustained increase of the resting membrane potential in cerebellar granule cells cultured either in high or low KCl-containing medium, and this long-term depolarizing effect of PACAP was mimicked by the I(K) specific blocker tetraethylammonium chloride (TEA). In addition, pre-incubation of granule cells with TEA suppressed the effect of PACAP on resting membrane potential. TEA mimicked the neuroprotective effect of PACAP against ethanol-induced apoptotic cell death, and the increase of caspase-3 activity observed after exposure of granule cells to ethanol was also significantly inhibited by TEA. Taken together, the present results demonstrate that, in rat cerebellar granule cells, PACAP reduces the delayed outward rectifier K(+) current by activating a type 1 PACAP (PAC1) receptor coupled to the adenylyl cyclase/protein kinase A pathway through a cholera toxin-sensitive Gs protein. Our data also show that PACAP and TEA induce long-term depolarization of the resting membrane potential, promote cell survival and inhibit caspase-3 activity, suggesting that PACAP-evoked inhibition of I(K) contributes to the anti-apoptotic effect of the peptide on cerebellar granule cells.

4-Aminopyridine↗

PubMed2002

Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons.

Voltage-gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0-3) and in 85-90 % of the neurons from older animals (postnatal days 21-60). In voltage clamp studies, alpha-dendrotoxin (alpha-DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K(+) currents of the sensory neurons. alpha-DTX blocked current in both A- and C-type neurons. The current had characteristics of a delayed rectifier with activation positive to -50 mV and little inactivation during 250 ms pulses. In current-clamp experiments alpha-DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A- and C-type neurons. However, there were prominent effects on excitability. alpha-DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after-hyperpolarization and increased the frequency/pattern of discharge of A- and C-type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K(+) currents function in response to the loss of the alpha-DTX-sensitive current to effect these changes in action potential wave shape and discharge.

Algorithms↗

PubMed2002

Effects of NIP-141 on K currents in human atrial myocytes.

A novel benzopyran derivative, NIP-141, effectively terminates experimental atrial fibrillation in canine hearts by prolonging atrial refractoriness. However, the effects of this drug on human atrial myocytes are unknown. This experiment evaluated the effects of NIP-141 on K currents in isolated human atrial myocytes using a whole-cell voltage-clamp method. NIP-141 inhibited the transient outward current (I(to)) and the ultra-rapid delayed rectifier K current (I(Kur)), each in a dose-dependent manner, with half-maximal inhibition concentrations of 16.3 microM and 5.3 microM, respectively (n = 5). NIP-141 inhibited both K currents in a voltage- and use-independent fashion, and it preferentially blocked them in the open state and dissociated rapidly from the channel. Because both K currents contribute significantly to the repolarization of the atrial action potential, these findings suggest that NIP-141 may terminate atrial fibrillation by prolonging action potential duration.

Action Potentials↗

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↗