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Results for “Kv1.2 Potassium Channel”

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PubMed2000

Abnormal axonal physiology is associated with altered expression and distribution of Kv1.1 and Kv1.2 K+ channels after chronic spinal cord injury.

Dysfunction of surviving axons which traverse the site of spinal cord injury (SCI) has been linked to altered sensitivity to the K+ channel blocker 4-aminopyridine (4-AP) and appears to contribute to post-traumatic neurological deficits although the underlying mechanisms remain unclear. In this study, sucrose gap electrophysiology in isolated dorsal column strips, Western blotting and confocal immunofluorescence microscopy were used to identify the K+ channels associated with axonal dysfunction after chronic (6-8 weeks postinjury) clip compresssion SCI of the thoracic cord at T7 in rats. The K+ channel blockers 4-AP (200 microM, 1 mM and 10 mM) and alpha-dendrotoxin (alpha-DTX, 500 nM) resulted in a significant relative increase in the amplitude and area of compound action potentials (CAP) recorded from chronically injured dorsal column axons in comparison with control noninjured preparations. In contrast, TEA (10 mM) and CsCl (2 mM) had similar effects on injured and control spinal cord axons. Western blotting and quantitative immunofluorescence microscopy showed increased expression of Kv1.1 and Kv1.2 K+ channel proteins on spinal cord axons following injury. In addition, Kv1.1 and Kv1.2 showed a dispersed staining pattern along injured axons in contrast to a paired juxtaparanodal localization in uninjured spinal cord axons. Furthermore, labelled alpha-DTX colocalized with Kv1.1 and Kv1.2 along axons. These findings suggest a novel mechanism of axonal dysfunction after SCI whereby an increased 4-AP- and alpha-DTX-sensitive K+ conductance, mediated in part by increased Kv1.1 and Kv1.2 K+ channel expression, contributes to abnormal axonal physiology in surviving axons.

4-Aminopyridine↗

PubMed2004

Calnexin regulates mammalian Kv1 channel trafficking.

Voltage-gated Kv1 channels are key factors regulating excitability in the mammalian central nervous system. Diverse posttranslational regulatory mechanisms operate to determine the density, subunit composition, and localization of Kv1 channel complexes in the neuronal plasma membrane. In this study, we investigated the role of the endoplasmic reticulum chaperone calnexin in the intracellular trafficking of Kv1 channels. We found that coexpressing calnexin with the Kv1.2alpha subunit in transfected mammalian COS-1 cells produced a dramatic dose-dependent increase in cell surface Kv1.2 channel complexes. In calnexin-transfected COS-1 cells, the proportion of Kv1.2 channels with mature N-linked oligosaccharide chains was comparable to that observed in neurons. In contrast, calnexin coexpression exerted no effects on trafficking of the intracellularly retained Kv1.1 or Kv1.6alpha subunits. We also found that calnexin and auxiliary Kvbeta2 subunit coexpression was epistatic, suggesting that they share a common pathway for promoting Kv1.2 channel surface expression. These results provide yet another component in the elaborate repertoire of determinants regulating the density of Kv1 channels in the plasma membrane.

Animals↗

PubMed2003

Presynaptic rat Kv1.2 channels suppress synaptic terminal hyperexcitability following action potential invasion.

Voltage-gated K+ channels activating close to resting membrane potentials are widely expressed and differentially located in axons, presynaptic terminals and cell bodies. There is extensive evidence for localisation of Kv1 subunits at many central synaptic terminals but few clues to their presynaptic function. We have used the calyx of Held to investigate the role of presynaptic Kv1 channels in the rat by selectively blocking Kv1.1 and Kv1.2 containing channels with dendrotoxin-K (DTX-K) and tityustoxin-Kalpha (TsTX-Kalpha) respectively. We show that Kv1.2 homomers are responsible for two-thirds of presynaptic low threshold current, whilst Kv1.1/Kv1.2 heteromers contribute the remaining current. These channels are located in the transition zone between the axon and synaptic terminal, contrasting with the high threshold K+ channel subunit Kv3.1 which is located on the synaptic terminal itself. Kv1 homomers were absent from bushy cell somata (from which the calyx axons arise); instead somatic low threshold channels consisted of heteromers containing Kv1.1, Kv1.2 and Kv1.6 subunits. Current-clamp recording from the calyx showed that each presynaptic action potential (AP) was followed by a depolarising after-potential (DAP) lasting around 50 ms. Kv1.1/Kv1.2 heteromers had little influence on terminal excitability, since DTX-K did not alter AP firing. However TsTX-Kalpha increased DAP amplitude, bringing the terminal closer to threshold for generating an additional AP. Paired pre- and postsynaptic recordings confirmed that this aberrant AP evoked an excitatory postsynaptic current (EPSC). We conclude that Kv1.2 channels have a general presynaptic function in suppressing terminal hyperexcitability during the depolarising after-potential.

Action Potentials↗

PubMed1995

Differential distribution of closely related potassium channels in rat Schwann cells.

Closely related K+ channels can coassemble to form heteromultimers in expression systems, as well as in vivo. Whether in vivo this coassembly is random and inevitable or whether highly homologous channels can be segregated and targeted independently within a given cell has not been determined. In this study, we address these questions by characterizing and localizing voltage-dependent K+ channels in Schwann cells. Transcripts for three closely related members of the Shaker-like family of K+ channels are found in adult rat sciatic nerve: Kv1.1, Kv1.2, and Kv1.5. We have examined two of these and observed that both Kv1.1 and Kv1.5 proteins are expressed in Schwann cells but differ in their distributions. Kv1.5 is localized on the Schwann cell membrane at the nodes of Ranvier and in bands that run along the outer surface of the myelin. It is also seen intracellularly in the vicinity of the nucleus. Schwann cell staining for Kv1.1, on the other hand, was seen only in perinuclear, intracellular compartments. These results provide evidence that closely related channels from the same family need not coassemble and can be localized differentially in the same cell. In addition, Kv1.1 was highly concentrated in the axonal membrane at juxtaparanodal regions. The distributions of these K+ channels in myelinated nerve highlight the elaborate molecular specializations of these membranes.

Animals↗

PubMed1997

Heterogeneity in K+ channel transcript expression detected in isolated ferret cardiac myocytes.

The molecular basis of the potassium ion (K+) channels that generate repolarization in heart tissue remains uncertain, in part because of the molecular diversity of the voltage-gated K+ channel family. In our investigation, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes to determine the identity, regional distribution, and cellular distribution of voltage-gated K+ channel, alpha-subunit mRNA expressed in ferret heart. The regions studied were from the sinoatrial node (SA), right and left atrium, right and left ventricle, and interatrial and interventricular septa. Kv1.5 and Kv1.4 were the most widely distributed K+ channel transcripts in the ferret heart (present in approximately 70%-86% and approximately 46%-95% of tested myocytes, respectively), followed by Kv1.2, Kv2.1, and Kv4.2. In addition, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family. Kv1.1, Kv1.6, and Kv6.1 were rarely expressed in working myocytes, but were more commonly expressed in SA nodal cells. Two other transcripts whose genes have been implicated in the long QT syndrome, erg and KvLQT1, were common in all regions (approximately 41%-58% and 52%-72%, respectively). These results show that both the diversity and heterogeneity of K+ channel mRNA in heart tissue is greater than previously suspected.

Animals↗

PubMed2003

Voltage-gated K+ channels in rat small cerebral arteries: molecular identity of the functional channels.

Voltage-gated potassium (KV) channels represent an important dilator influence in the cerebral circulation, but the composition of these tetrameric ion channels remains unclear. The goals of the present study were to evaluate the contribution of KV1 family channels to the resting membrane potential and diameter of small rat cerebral arteries, and to identify the alpha-subunit composition of these channels using patch-clamp, molecular and immunological techniques. Initial studies indicated that 1 micromol l(-1) correolide (COR), a specific antagonist of KV1 channels, depolarized vascular smooth muscle cells (VSMCs) in pressurized (60 mmHg) cerebral arteries from -55 +/- 1 mV to -34 +/- 1 mV, and reduced the resting diameter from 152 +/- 15 microm to 103 +/- 20 microm. In patch clamped VSMCs from these arteries, COR-sensitive KV1 current accounted for 65 % of total outward KV current and was observed at physiological membrane potentials. RT-PCR identified mRNA encoding each of the six classical KV1 alpha-subunits, KV1.1-1.6, in rat cerebral arteries. However, only the KV1.2 and 1.5 proteins were detected by Western blot. The expression of these proteins in VSMCs was confirmed by immunocytochemistry and co-immunoprecipitation of KV1.2 and 1.5 from VSMC membranes suggested KV1.2/1.5 channel assembly. Subsequently, the pharmacological and voltage-sensitive properties of KV1 current in VSMCs were found to be consistent with a predominant expression of KV1.2/1.5 heterotetrameric channels. The findings of this study suggest that KV1.2/1.5 heterotetramers are preferentially expressed in rat cerebral VSMCs, and that these channels contribute to the resting membrane potential and diameter of rat small cerebral arteries.

Animals↗

PubMed2001

Kv beta subunit oxidoreductase activity and Kv1 potassium channel trafficking.

Voltage-gated Kv1 potassium channels consist of pore-forming alpha subunits and cytoplasmic Kv beta subunits. The latter play diverse roles in modulating the gating, stability, and trafficking of Kv1 channels. The crystallographic structure of the Kv beta2 subunit revealed surprising structural homology with aldo-keto reductases, including a triosephosphate isomerase barrel structure, conservation of key catalytic residues, and a bound NADP(+) cofactor (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell 90, 943-952). Each Kv1-associated Kv beta subunit (Kv beta 1.1, Kv beta 1.2, Kv beta 2, and Kv beta 3) shares striking amino acid conservation in key catalytic and cofactor binding residues. Here, by a combination of structural modeling and biochemical and cell biological analyses of structure-based mutations, we investigate the potential role for putative Kv beta subunit enzymatic activity in the trafficking of Kv1 channels. We found that all Kv beta subunits promote cell surface expression of coexpressed Kv1.2 alpha subunits in transfected COS-1 cells. Kv beta1.1 and Kv beta 2 point mutants lacking a key catalytic tyrosine residue found in the active site of all aldo-keto reductases have wild-type trafficking characteristics. However, mutations in residues within the NADP(+) binding pocket eliminated effects on Kv1.2 trafficking. In cultured hippocampal neurons, Kv beta subunit coexpression led to axonal targeting of Kv1.2, recapitulating the Kv1.2 localization observed in many brain neurons. Similar to the trafficking results in COS-1 cells, mutations within the cofactor binding pocket reduced axonal targeting of Kv1.2, whereas those in the catalytic tyrosine did not. Together, these data suggest that NADP(+) binding and/or the integrity of the binding pocket structure, but not catalytic activity, of Kv beta subunits is required for intracellular trafficking of Kv1 channel complexes in mammalian cells and for axonal targeting in neurons.

Animals↗

PubMed1999

Heterogeneous expression of voltage-gated potassium channels of the shaker family (Kv1) in oligodendrocyte progenitors.

Outwardly rectifying K(+) channels determine the membrane conductance and influence the proliferation rate of glial progenitor cells. To analyze the molecular identity and the functional role of K(+) channels in glial progenitors of mouse brain, expression of shaker-type Kv1 genes was studied at three levels: (1) presence of Kv1 mRNAs, (2) biosynthesis of channel proteins and (3) electrophysiological and pharmacological properties of K(+) currents. mRNA expression of Kv1.1 to Kv1.6 genes was studied by single-cell reverse transcription-mediated polymerase chain reaction (RT-PCR) using degenerate primers to amplify the six Kv1 transcripts. Most cells expressed several mRNA combinations simultaneously. In more than half of the cells, messages for Kv1.2, Kv1.5 and Kv1.6 were found, while Kv1.1, Kv1.3 and Kv1.4 were detected in only a minority of cells. In contrast, at the level of protein expression - employing immunocytochemistry with subtype-specific antibodies - Kv1. 2 and Kv1.3 were undetectable (<2%), while almost all cells expressed Kv1.4 (85%), Kv1.5 (99%) and Kv1.6 (99%). Kv1.1 was present in a minor cell population (10%). Functional contribution of Kv1 proteins to progenitor membrane conductance was determined by analyzing the voltage-dependence of K(+) current activation and inactivation as well as their current sensitivities to the subtype-preferring blockers and toxins tetraethylammonium (TEA), 4-aminopyridine (4-AP), charybdotoxin (CTX), alpha-dendrotoxin (DTX) and mast-cell degranulating peptide (MCDP). From these results, it is concluded: first, glial progenitor cells can express all transcripts of the six Kv1 genes, but do not express all proteins; second, Kv1.4, Kv1.5 and Kv1.6 proteins are most abundant and were found in the majority of cells; and third, K(+) currents flow predominantly either through heteromeric channel complexes or through homomeric Kv1.5 ion pores, but not through homomeric Kv1.4 or Kv1.6 channels.

Animals↗

PubMed2001

Voltage-gated K+ channels in chemoreceptor sensory neurons of rat petrosal ganglion.

A subpopulation of sensory neurons in the petrosal ganglion transmits information between peripheral chemoreceptors (glomus cells) in the carotid body and relay neurons in the nucleus of the solitary tract. Expression of voltage-gated K+ channels in these neurons was characterized by immunohistochemical localization. Five members of the Kv1 family, Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 and members of two other families, Kv2.1 and Kv4.3, were identified in over 90% of the chemoreceptor neurons. Although the presence of these channel proteins was consistent throughout the population, individual neurons showed considerable variation in K+ current profiles.

Animals↗

PubMed2001

Age-related changes in the distribution of Kv1.1 and Kv1.2 channel subunits in the rat cerebellum.

We have revealed age-related changes in the expression patterns of Kv1.1 and Kv1.2 in the rat cerebellum for the first time. In the aged rat, immunoreactivity for Kv1.1 was increased in the cell bodies of Purkinje cells, while the staining intensity was significantly decreased in the granule cells. The cell bodies of cerebellar output neurons showed strong Kv1.1 immunoreactivity in the nucleus medialis, interpositus and lateralis of the aged rat. Kv1.2 immunoreactivity was found in some interneurons with their processes in this region of the aged rat. Image analysis demonstrated that immunoreactivities for Kv1.1 and Kv1.2 were increased specifically in the cell bodies of cerebellar output neurons of the aged rat. This study may provide useful data for future investigations on the channels that cause brain diseases and age-related disorders.

Aging↗

PubMed2001

Immunohistochemical study on the distribution of six members of the Kv1 channel subunits in the rat cerebellum.

Voltage-gated K(+) (Kv) channels are critical for a wide variety of processes, and play an essential role in neurons. In the present study, we have demonstrated a unique pattern of expression of the six Kv1 channel subunits in the rat cerebellum, for the first time. The greatest concentration of Kv1.2 was found in the basket cell axon plexus and terminal regions around the Purkinje cells. Relatively weak immunoreactivity for Kv1.1 was also found in this area. The somatodendritic Purkinje cell areas were intensely stained with anti-Kv1.5 antibodies. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis. Interestingly, Kv1.2 immunoreactivity was found in some neurons with their processes. Our immunohistochemical results may support the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the cerebellum.

Animals↗

PubMed1999

Recreation of neuronal Kv1 channel oligomers by expression in mammalian cells using Semliki Forest virus.

The multiple roles of voltage-sensitive K(+) channels (Kv1 subfamily) in brain are served by subtypes containing pore-forming alpha (1.1-1.6) and auxiliary beta subunits, usually in an (alpha)(4)(beta)(4) stoichiometry. To facilitate structure/activity analysis, combinations that are prevalent in neurones and susceptible to alpha-dendrotoxin (alphaDTX) were reproduced in mammalian cells, using Semliki Forest virus. Infected Chinese hamster ovary cells expressed N-glycosylated Kv1.1 and 1.2 alpha subunits (M(r) approximately 60 and 62 K) that assembled and bound [(125)I]-alphaDTX with high affinity; an appreciable proportion appeared on the cell surface, with Kv1.2 showing a 5-fold enrichment in a plasma membrane fraction. To obtain 'native-like' alpha/beta complexes, beta1.1 or 2.1 (M(r) approximately 42 and 39 K, respectively) was co-expressed with Kv1.1 or 1.2. This slightly enhanced N-glycosylation and toxin binding, most notable with beta2. 1 and Kv1.2. Solubilization of membranes from cells infected with Kv. 1.2 and beta2.1, followed by Ni(2+) chromatography, gave a purified alpha1.2/beta2.1 complex with a size of approximately 405 K and S(20, W) = 15.8 S. Importantly, these values indicate that four alpha and beta subunits co-assembled as in neurones, a conclusion supported by the size ( approximately 260 K) of the homo-tetramer formed by Kv1.2 alone. Thus, an authentic K(+) channel octomer has been reconstructed; oligomeric species were also found in plasma membranes. To create 'authentic-like' hetero-oligomeric channels, Kv1.1 and 1.2 were co-expressed and shown to have assembled by the precipitation of both with IgGs specific for either. Consistently, confocal microscopy of cells labeled with these antibodies showed that the relatively low surface content of Kv1.1 was increased by Kv1.2. [(125)I]-alphaDTX binding to these complexes was antagonized by DTX(k), a probe selective for Kv1.1, in a manner that mimicks the pattern observed for the Kv1.1/1.2-containing channels in neuronal membranes.

Animals↗

PubMed2004

Temporal and spatial patterns of Kv1.1 and Kv1.2 protein and gene expression in spinal cord white matter after acute and chronic spinal cord injury in rats: implications for axonal pathophysiology after neurotrauma.

After spinal cord injury (SCI), surviving white matter axons display axonal dysfunction associated with demyelination and altered K+ channel activity. To clarify the molecular basis of posttraumatic axonal pathophysiology after SCI, we investigated the changes in expression and distribution of the axonal K+ channel subunits Kv1.1 and Kv1.2 in spinal cord white matter after in vivo SCI in the rat. Using Western blot analysis, we found an increased expression of Kv1.1 and Kv1.2 at 2 and 6 weeks after SCI. By real-time PCR we observed an increase in Kv1.1 and Kv1.2 mRNA levels 1 day after SCI, which persisted until 6 weeks. Confocal immunohistochemistry showed a markedly dispersed labelling of Kv1.1 and Kv1.2 along the injured axons, in contrast to the tight localization of these channels to the juxtaparanodes of noninjured axons. This redistribution of Kv1.1 and Kv1.2 occurred as early as 1 h postinjury along some injured axons, and persisted at 6 weeks postinjury. In parallel with the redistribution of Kv1.1 and 1.2, contactin-associated protein (Caspr), which is normally confined to a paranodal location, also displayed a more diffuse distribution along the injured spinal cord axons. Our results suggest that the increased expression of Kv1.1 and Kv1.2 proteins is transcriptionally regulated. In contrast, the redistribution of the axonal K+ channel subunits occurs very early postinjury and probably reflects a disruption of the juxtaparanodal axonal region due to physical trauma, as shown by altered localization of Caspr.

Actins↗

PubMed2004

Evidence for domain-specific recognition of SK and Kv channels by MTX and HsTx1 scorpion toxins.

Maurotoxin (MTX) and HsTx1 are two scorpion toxins belonging to the alpha-KTx6 structural family. These 34-residue toxins, cross-linked by four disulfide bridges, share 59% sequence identity and fold along the classical alpha/beta scaffold. Despite these structural similarities, they fully differ in their pharmacological profiles. MTX is highly active on small (SK) and intermediate (IK) conductance Ca(2+)-activated (K(+)) channels and on voltage-gated Kv1.2 channel, whereas HsTx1 potently blocks voltage-gated Kv1.1 and Kv1.3 channels only. Here, we designed and chemically produced MTX-HsTx1, a chimera of both toxins that contains the N-terminal helical region of MTX (sequence 1-16) and the C-terminal beta-sheet region of HsTx1 (sequence 17-34). The three-dimensional structure of the peptide in solution was solved by (1)H NMR. MTX-HsTx1 displays the activity of MTX on SK channel, whereas it exhibits the pharmacological profile of HsTx1 on Kv1.1, Kv1.2, Kv1.3, and IK channels. These data demonstrate that the helical region of MTX exerts a key role in SK channel recognition, whereas the beta-sheet region of HsTx1 is crucial for activity on all other channel types tested.

Amino Acid Sequence↗

PubMed2002

Developmental changes in the functional characteristics and expression of voltage-gated K+ channel currents in rat aortic myocytes.

OBJECTIVE: Active control of the arterial diameter by vascular smooth muscle is one of the principle mechanisms by which vessels adapt to a significant rise in blood pressure after birth. Although voltage-gated K+ (Kv) channels play an important role in the regulation of excitation-contraction coupling in arteries, very little is known about postnatal modification of Kv channels. We therefore investigated changes in the functional characteristics and expression of Kv channels in rat aortic myocytes (RAMs) during early postnatal development. METHODS: Kv currents (I(Kv)) were investigated in single smooth muscle cells freshly dispersed from neonatal (1-3 days) and adult Wistar rat thoracic aorta using the whole-cell patch clamp technique. RESULTS: I(Kv) in neonates had significantly faster activation kinetics and was inactivated at more positive voltages than I(Kv) in adults (half-inactivation potential -24+/-2 and -40+/-3 mV and slope factor 4.2+/-0.4 and 11.1+/-0.5 mV, respectively). No difference in the steady state activation was found. I(Kv) in neonates was insensitive to a high concentration of tetraethylammonium (TEA, 10 mM) but blocked 4-aminopyridine (4-AP, IC(50)=0.5+/-0.1 mM), whereas I(Kv) in adult RAMs was almost completely abolished by 10 mM TEA and was relatively insensitive to low concentrations of 4-AP. I(Kv) in both age groups was insensitive to charybdotoxin (300 nM) or alpha-dendrotoxin (200 nM). Immunoblot analysis showed that the expression of Kv1.2 alpha-protein decreased and Kv2.1 increased with development. CONCLUSION: Significant changes in functional characteristics of the native I(Kv) and the expression of particular Kv channel proteins occurred during postnatal vascular development. These changes could play an important role in adaptation to extrauterine life.

4-Aminopyridine↗

PubMed2004

Heteromultimeric Kv1 channels contribute to myogenic control of arterial diameter.

Inhibition of vascular smooth muscle (VSM) delayed rectifier K+ channels (K(DR)) by 4-aminopyridine (4-AP; 200 micromol/L) or correolide (1 micromol/L), a selective inhibitor of Kv1 channels, enhanced myogenic contraction of rat mesenteric arteries (RMAs) in response to increases in intraluminal pressure. The molecular identity of K(DR) of RMA myocytes was characterized using RT-PCR, real-time PCR, and immunocytochemistry. Transcripts encoding the pore-forming Kvalpha subunits, Kv1.2, Kv1.4, Kv1.5, and Kv1.6, were identified and confirmed at the protein level with subunit-specific antibodies. Kvbeta transcript (beta1.1, beta1.2, beta1.3, and beta2.1) expression was also identified. Kv1.5 message was approximately 2-fold more abundant than that for Kv1.2 and Kv1.6. Transcripts encoding these three Kv1alpha subunits were approximately 2-fold more abundant in 1st/2nd order conduit compared with 4th order resistance RMAs, and Kvbeta1 was 8-fold higher than Kvbeta2 message. RMA K(DR) activated positive to -50 mV, exhibited incomplete inactivation, and were inhibited by 4-AP and correolide. However, neither alpha-dendrotoxin or kappa-dendrotoxin affected RMA K(DR), implicating the presence of Kv1.5 in all channels and the absence of Kv1.1, respectively. Currents mediated by channels because of coexpression of Kv1.2, Kv1.5, Kv1.6, and Kvbeta1.2 in human embryonic kidney 293 cells had biophysical and pharmacological properties similar to those of RMA K(DR). It is concluded that K(DR) channels composed of heteromultimers of Kv1 subunits play a critical role in myogenic control of arterial diameter.

4-Aminopyridine↗

PubMed2003

Kv channel subunits that contribute to voltage-gated K+ current in renal vascular smooth muscle.

The rat renal arterial vasculature displays differences in K(+) channel current phenotypes along its length. Small arcuate to cortical radial arteries express a delayed rectifier phenotype, while the predominant Kv current in larger arcuate and interlobar arteries is composed of both transient and sustained components. We sought to determine whether Kvalpha subunits in the rat renal interlobar and arcuate arteries form heterotetramers, which may account for the unique currents, and whether modulatory Kvbeta subunits are present in renal vascular smooth muscle cells. RT-PCR indicated the presence of several different Kvalpha subunit isoform transcripts. Co-immunoprecipitation with immunoblotting and immunohistochemical evidence suggests that a portion of the K(+) current phenotype is a heteromultimer containing delayed-rectifier Kv1.2 and A-type Kv1.4 channel subunits. RT-PCR and immunoblot analyses also demonstrated the presence of both Kvbeta1.2 and Kvbeta1.3 in renal arteries. These results suggest that heteromultimeric formation of Kvalpha subunits and the presence of modulatory Kvbeta subunits are important factors in mediating Kv currents in the renal microvasculature and suggest a potentially critical role for these channel subunits in blood pressure regulation.

Animals↗

PubMed1998

Activation of protein tyrosine kinase PYK2 by the m1 muscarinic acetylcholine receptor.

Several G protein-coupled receptors are known to direct the tyrosine phosphorylation, and in some cases the activation, of diverse tyrosine kinases. Using a stable cell line approach, we characterize the activation of PYK2, a tyrosine kinase structurally related to focal adhesion kinase, by the G protein-coupled m1 muscarinic acetylcholine receptor. We find that PYK2 tyrosine kinase activity is critical for the m1 receptor-stimulated tyrosine phosphorylation of PYK2. Furthermore, we identify two tyrosine residues that are subject to phosphorylation in response to muscarinic signaling and show that this phosphorylation induces two cytosolic proteins, c-Src and Grb2, to bind to PYK2. This is the first demonstration of the significance played by distinct PYK2 tyrosine residues in G protein-coupled signaling to this kinase. By comparison, though m1 receptors induce the tyrosine phosphorylation of the cytoskeletal protein paxillin, the association of paxillin with PYK2 is unaffected by muscarinic signaling. We also provide evidence that PYK2 specifically phosphorylates the carboxyl-terminal cytosolic portion of the potassium channel Kv1.2 in a manner regulated by the m1 receptor. These results delineate molecular events attending the m1 muscarinic receptor stimulation of this tyrosine kinase and establish PYK2 as an effector of the m1 muscarinic receptor in the regulation of multiple cell functions.

Adaptor Proteins, Signal Transducing↗