Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A

Protein kinase A (PKA) stimulates Cl secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a tightly regulated Cl- channel in the apical membrane of many secretory epithelia. The CFTR channel is also modulated by protein kinase C (PKC), but the regulatory mechanisms are poorly understood. Here we present evidence that PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA. When patches were excised from transfected Chinese hamster ovary cells, CFTR responses to PKA became progressively smaller with time and eventually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PKC and was reversed by the addition of PKC to channels that had become refractory to PKA. PKC enhanced PKA stimulation of open probability without increasing the number of functional channels. Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 microM) reduced the channel activity that could be elicited by forskolin in cell-attached patches. Moreover, in whole cell patches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PKC inhibitors, although an abrupt, partial activation was observed after a delay of >15 min. Modulation by PKC was most pronounced when basal PKC phosphorylation was reduced by briefly preincubating cells with chelerythrine. Constitutive PKC phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is approximately 60% of that attained during in vitro exposure to both kinases. Differences in basal PKC activity may contribute to the variable cAMP responsiveness of CFTR channels in different cell types.     

Protein kinase C-mediated regulation of L-type Ca channels from skeletal muscle requires phosphorylation of the alpha 1 subunit

Dihydropyridine-sensitive, L-type Ca channels are hetero-oligomeric proteins that are modulated in certain cell types by protein kinase C (PKC). In native skeletal muscle membranes, PKC phosphorylates the alpha 1 and beta subunits of these Ca channels and modulates channel activity. However, it is unknown if phosphorylation of both subunits is necessary for PKC-mediated channel regulation. Here we report that stoichiometric phosphorylation of the alpha 1 subunit was required for activation of these Ca channels by PKC, while PKC-mediated phosphorylation of the beta subunit alone did not modify channel activity. Furthermore, reversal of the functional effects of PKC by protein phosphatase-1c was quantitatively correlated with dephosphorylation of the alpha 1 subunit.     

Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C

Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.     

Regulation of IRK3 inward rectifier K+ channel by m1 acetylcholine receptor and intracellular magnesium

Inward rectifier K+ channels control the cell's membrane potential and neuronal excitability. We report that the IRK3 but not the IRK1 inward rectifier K+ channel activity is inhibited by m1 muscarinic acetylcholine receptor. This m1 modulation cannot be accounted for by protein kinase C, Ca2+, or channel phosphorylation, but can be mimicked by Mg2+. Based on quantitative analyses of IRK3 and two different IRK1 mutant channels bestowed with sensitivity to m1 modulation, we suggest that the resting Mg2+ level causes chronic inhibition of IRK3 channels, and m1 receptor stimulation may lead to an increase of cytoplasmic Mg2+ concentration and further channel inhibition, due to the ability of Mg2+ to lead these channels into a prolonged inactivated state.     

Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.     

Modulation of rod photoreceptor cyclic nucleotide-gated channels by tyrosine phosphorylation

Cyclic nucleotide-gated (CNG) channels in vertebrate photoreceptors are crucial for transducing light-induced changes in cGMP concentration into electrical signals. In this study, we show that both native and exogenously expressed CNG channels from rods are modulated by tyrosine phosphorylation. The cGMP sensitivity of CNG channels, composed of rod alpha-subunits expressed in Xenopus oocytes, gradually increases after excision of inside-out patches from the oocyte membrane. This increase in sensitivity is inhibited by a protein tyrosine phosphatase (PTP) inhibitor and is unaffected by three different Ser/Thr phosphatase inhibitors. Moreover, it is suppressed or reversed by application of ATP but not by a nonhydrolyzable ATP analog. Application of protein tyrosine kinase (PTK) inhibitors causes an increase in cGMP sensitivity, but only in the presence of ATP. Taken together, these results suggest that CNG channels expressed in oocytes are associated with active PTK(s) and PTP(s) that regulate their cGMP sensitivity by changing phosphorylation state. The cGMP sensitivity of native CNG channels from salamander rod outer segments also increases and decreases after incubation with inhibitors of PTP(s) and PTK(s), respectively. These results suggest that rod CNG channels are modulated by tyrosine phosphorylation, which may function as a novel mechanism for regulating the sensitivity of rods to light.     

Alterations of potassium channel activity in retinal Müller glial cells induced by arachidonic acid

Arachidonic acid, which is thought to be involved in pathogenetic mechanisms of the central nervous system, has been shown previously to modulate neuronal ion channels and the glutamate uptake carrier of retinal glial (Müller) cells. We have used various configurations of the patch-clamp technique to determine the effects of arachidonic acid on the K+ currents of freshly isolated Müller glial cells from rabbit and human. Arachidonic acid reduced the peak amplitude of the transient (A-type) outward K+ currents in a dose-dependent and reversible manner, with a 50% reduction achieved by 4.1 microM arachidonic acid. The inward rectifier-mediated currents remained unchanged after arachidonic acid application. The amplitude of the Ca(2+)-activated K+ outward currents (KCa), which were blocked by 1 mM tetraethylammonium chloride and 40 nM iberiotoxin, respectively, was dose-dependently elevated by bath application of arachidonic acid. The activation curve of the KCa currents shifted towards more negative membrane potentials. Furthermore, arachidonic acid was found to suppress inwardly directed Na+ currents. In cell-attached recordings with 3 mM K+ in the bath and 130 mM K+ in the pipette, the KCa channels of rabbit Müller cells displayed a linear current-voltage relation, with a mean slope conductance of 102 pS. In excised patches, the slope conductance was 220 pS (150 mM K+i/130 mM K+o). The opening probability of the KCa channels increased during membrane depolarization and during elevation of the free Ca2+ concentration at the intracellular face of the membrane patches. Bath application of arachidonic acid caused a reversible increase of the single-channel opening probability, as well as an increase of the number of open channels. Arachidonic acid did not affect the single-channel conductance. Since arachidonic acid also stimulates the KCa channel activity in excised patches, the action of arachidonic acid is assumed to be independent of changes of the intracellular calcium concentration. Our results demonstrate that arachidonic acid exerts specific effects on distinct types of K+ channels in retinal glial, cells. In pathological cases, elevated arachidonic acid levels may contribute to prolonged Müller cell depolarizations, and to the initiation of reactive glial cell proliferation.     

In vivo whole-cell recording from neurons of the superior colliculus in fetal rats

The actions of halothane on serotonin-sensitive potassium channels (S K+ channels) were studied in sensory neurons of Aplysia. The normalized open probability of S K+ channels was increased by clinical concentrations of halothane in cell-attached and excised patches from neurons of the pleural ventrocaudal cluster. No voltage-dependence of channel activation by halothane was observed. Pre-treatment of neurons with 8-bromo-cAMP (8-Br-cAMP) or nordihydroguaiaretic acid (NDGA) had no effect on the relative level of channel activation by halothane. S K+ channels that were activated by arachidonic acid could also be activated by halothane and exhibited closely similar amplitude distributions of open channel current. Results from these experiments showed that S K+ channel activation by halothane did not depend on second messenger modulation of channel activity. We conclude that it is likely that halothane directly activates S K+ channels.     

Protein kinase C disrupts cannabinoid actions by phosphorylation of the CB1 cannabinoid receptor

We have found that phosphorylation of a G-protein-coupled receptor by protein kinase C (PKC) disrupts modulation of ion channels by the receptor. In AtT-20 cells transfected with rat cannabinoid receptor (CB1), the activation of an inwardly rectifying potassium current (Kir current) and depression of P/Q-type calcium channels by cannabinoids were prevented by stimulation of protein kinase C by 100 nM phorbol 12-myristate 13-acetate (PMA). In contrast, activation of Kir current by somatostatin was unaffected, and inhibition of calcium channels was only modestly attenuated. The possibility that PKC acted by phosphorylating CB1 receptors was confirmed by demonstrating that PKC phosphorylated a single serine (S317) of a fusion protein incorporating the third intracellular loop of CB1. Mutating this serine to alanine did not affect the ability of CB1 to modulate currents, but it eliminated disruption by PMA, demonstrating that PKC can disrupt ion channel modulation by receptor phosphorylation.     

Calcium modulation of ligand affinity in the cyclic GMP-gated ion channels of cone photoreceptors

To investigate modulation of the activation of cGMP-gated ion channels in cone photoreceptors, we measured currents in membrane patches detached from the outer segments of single cones isolated from striped bass retina. The sensitivity of these channels to activation by cGMP depends on the history of exposure to divalent cations of the membrane's cytoplasmic surface. In patches maintained in 20 microM Ca++ and 100 microM Mg++ after excision, the current amplitude dependence on cGMP is well described by a Hill equation with average values of K1/2, the concentration necessary to activate half the maximal current, of 86 microM and a cooperativity index, n, of 2.57. Exposing the patch to a solution free of divalent cations irreversibly increases the cGMP sensitivity; the average value of K1/2 shifts to 58.8 microM and n shifts to 1.8. Changes in cGMP sensitivity do not affect other functional parameters of the ion channels, such as the interaction and permeation of mono- and divalent cations. Modulation of cGMP activation depends on the action of an endogenous factor that progressively dissociates from the channel as Ca++ concentration is lowered below 1 microM. The activity of the endogenous modulator is not well mimicked by exogenously added calmodulin, although this protein competes with the endogenous modulator for a common binding site. Thus, the modulation of cGMP affinity in cones depends on the activity of an unidentified molecule that may not be calmodulin.     

K+ channel modulation in arterial smooth muscle

Potassium channels play an essential role in the membrane potential of arterial smooth muscle, and also in regulating contractile tone. Four types of K+ channel have been described in vascular smooth muscle: Voltage-activated K+ channels (Kv) are encoded by the Kv gene family, Ca(2+)-activated K+ channels (BKCa) are encoded by the slo gene, inward rectifiers (KIR) by Kir2.0, and ATP-sensitive K+ channels (KATP) by Kir6.0 and sulphonylurea receptor genes. In smooth muscle, the channel subunit genes reported to be expressed are: Kv1.0, Kv1.2, Kv1.4-1.6, Kv2.1, Kv9.3, Kv beta 1-beta 4, slo alpha and beta, Kir2.1, Kir6.2, and SUR1 and SUR2. Arterial K+ channels are modulated by physiological vasodilators, which increase K+ channel activity, and vasoconstrictors, which decrease it. Several vasodilators acting at receptors linked to cAMP-dependent protein kinase activate KATP channels. These include adenosine, calcitonin gene-related peptide, and beta-adrenoceptor agonists. beta-adrenoceptors can also activate BKCa and Kv channels. Several vasoconstrictors that activate protein kinase C inhibit KATP channels, and inhibition of BKCa and Kv channels through PKC has also been described. Activators of cGMP-dependent protein kinase, in particular NO, activate BKCa channels, and possibly KATP channels. Hypoxia leads to activation of KATP channels, and activation of BKCa channels has also been reported. Hypoxic pulmonary vasoconstriction involves inhibition of Kv channels. Vasodilation to increased external K+ involves KIR channels. Endothelium-derived hyperpolarizing factor activates K+ channels that are not yet clearly defined. Such K+ channel modulations, through their effects on membrane potential and contractile tone, make important contributions to the regulation of blood flow.     

The large conductance, voltage-dependent, and calcium-sensitive K+ channel, Hslo, is a target of cGMP-dependent protein kinase phosphorylation in vivo

Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human pore-forming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by approximately 20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately 125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+ channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for PKG-Ialpha-induced activation of maxi-K channels.     

Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator

We demonstrate here that coexpression of ROMK2, an inwardly rectifying ATP-sensitive renal K+ channel (IKATP) with cystic fibrosis transmembrane regulator (CFTR) significantly enhances the sensitivity of ROMK2 to the sulfonylurea compound glibenclamide. When expressed alone, ROMK2 is relatively insensitive to glibenclamide. The interaction between ROMK2, CFTR, and glibenclamide is modulated by altering the phosphorylation state of either ROMK2, CFTR, or an associated protein, as exogenous MgATP and the catalytic subunit of protein kinase A significantly attenuate the inhibitory effect of glibenclamide on ROMK2. Thus CFTR, which has been demonstrated to interact with both Na+ and Cl- channels in airway epithelium, modulates the function of renal ROMK2 K+ channels.     

Differential regulation of N- and Q-type Ca2+ channels by cyclic nucleotides and G-proteins

Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway delimited to the membrane. In addition, modulation of Ca2+ channel activity by protein kinases also contributes to the dynamic regulation of neuronal physiology. Recently, differences in these modulations between Ca2+ channel subtypes have been shown in several neuronal preparations. Here we show that two types of presynaptic Ca2+ channel (N-type and Q-type) are differentially regulated by cAMP and G-proteins using a Xenopus oocyte expression system. Treatment to increase cytosolic cAMP concentration with forskolin and 3-isobutyl-1-methylxanthine (IBMX) markedly potentiated Q-type channel current, and the enhancement was reversed by protein kinase A inhibitors. Much smaller enhancement was observed in N-type channel current after the cAMP elevation. When large depolarizing prepulse was applied to the oocytes for evaluation of the tonic inhibition of Ca2+ channels by intrinsic G-protein activity, N-type channel current elicited a large prepulse facilitation but Q-type channels did not. The tonic inhibition of N-type channels was abolished by an intracellular perfusion with a 'cut-open' recording configuration, or by co-expression with G(alpha o). When kappa opioid receptors were co-expressed and stimulated with agonists, depolarization-resistant inhibition was more apparent in Q-type channels than in N-type channels. These results suggest that Q-type channels are more susceptible to the protein kinase A-mediated facilitation than N-type channels, and that activity of N-type channels can be more highly regulated in a voltage-dependent manner by G(betagamma) than that of Q-type channels. These differences may account for the selective regulation of neurotransmitter release by these Ca2+ channels.     

Clinical evaluation of the osteoarthritic patient

It is well-established that in heart, both the L-type Ca2+ channel and the cystic fibrosis transmembrane conductance regulator Cl- channel are regulated by cAMP-dependent phosphorylation. However, it is not clear whether both of these channels are regulated in concert by protein kinase A (PKA) or whether there are mechanisms that independently control the phosphorylation of these two PKA targets. The purpose of this study was to compare the effects of various protein phosphatase and protein kinase inhibitors on these two ionic currents (ICa and ICl) in guinea pig ventricular myocytes to gain insight into these questions. We found that both the stimulation and washout of the effects of isoproterenol on ICl are about twice as fast as the effects on ICa, probably because the dephosphorylation reaction for ICl is faster than that for ICa. In contrast, inhibition of protein phosphatases with 10 microM microcystin stimulated both ICa and ICl, but the stimulation of ICl was much slower and smaller than the stimulation of ICa. The effect of microcystin was inhibited by staurosporine (Ki = 171.5 and 161 nM for ICa and ICl, respectively), suggesting that the stimulation was due to a kinase. The kinase was not protein kinase C (PKC) because it was not inhibited by the specific pseudosubstrate inhibitor of PKC, PKC(19-31), and it was not PKA because it was not inhibited by adenosine 3',5'-cyclic phosphorothioate. These results suggest that although both the Ca2+ and Cl- channels are regulated by cAMP-dependent phosphorylation, another protein kinase may also regulate these channels, and the kinetics of the response of the channels to phosphorylation can be modulated independently by protein phosphatases.     

Reconstitution of a voltage and calcium dependent potassium channel from rat cerebellum

Membrane vesicles from rat cerebellum were reconstituted into lipid bilayers. The activity of two different potassium channels was recorded: (1) a small conducting voltage dependent potassium channel insensitive to [Ca2+]i, (2) a calcium and voltage dependent potassium channel (KCa). KCa channels had a conductance of (302+/-15) pS (n=5) and were activated by [Ca2+]i and membrane depolarizations. They were blocked by tetraethylamonium (TEA) and charybdotoxin (CTX) but insensitive to noxiustoxin (NTX). Finally, we showed the blocking effect of Androctonus australis Hector (AaH) scorpion venom on KCa channels from rat cerebellum.     

Protein kinase A-activated chloride channel is inhibited by the Ca(2+)-calmodulin complex in cardiac sarcoplasmic reticulum

Cardiac sarcoplasmic reticulum (SR) has several chloride (Cl-) channels, which may neutralize the charge across the SR membrane generated by Ca2+ movement. We recently reported a novel 116-picosiemen Cl- channel that is activated by protein kinase A-dependent phosphorylation in cardiac SR. This Cl- channel may serve as a target protein in the receptor-dependent regulation of cardiac excitation-contraction coupling. To understand further regulatory mechanisms, the effects of Ca2+ on the Cl- channel were studied using the planar lipid bilayer-vesicle fusion technique. In the presence of calmodulin (CaM, 0.1 mumol/L per microgram SR vesicles), Ca2+ (3 mumol/L to 1 mmol/L) added to the cis solution reduced the channel openings in a concentration-dependent fashion, whereas Ca2+ (1 nmol/L to 1 mmol/L) alone or CaM (0.1 to 1 mumol/L per microgram SR vesicles) with 1 nmol/L Ca2+ did not affect the channel activity. This inhibitory effect of Ca2+ in the presence of CaM was prevented by CaM inhibitors N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide and calmidazolium but not by CaM kinase II inhibitor KN62. These results suggest that the Ca(2+)-CaM complex itself, but not CaM kinase II, is involved in this channel inhibition. Thus, the cardiac SR 116-picosiemen Cl- channel is regulated not only by protein kinase A-dependent phosphorylation but also by the cytosolic Ca(2+)-CaM complex. This is a novel second messenger-mediated regulation of Cl- channels in cardiac SR membrane.     

Modulation of skeletal muscle sodium channels by human myotonin protein kinase

In myotonic muscular dystrophy, abnormal muscle Na currents underlie myotonic discharges. Since the myotonic muscular dystrophy gene encodes a product, human myotonin protein kinase, with structural similarity to protein kinases, we tested the idea that human myotonin protein kinase modulates skeletal muscle Na channels. Coexpression of human myotonin protein kinase with rat skeletal muscle Na channels in Xenopus oocytes reduced the amplitude of Na currents and accelerated current decay. The effect required the presence of a potential phosphorylation site in the inactivation mechanism of the channel. The mutation responsible for human disease, trinucleotide repeats in the 3' untranslated region, did not prevent the effect. The consequence of an abnormal amount of the kinase would be altered muscle cell excitability, consistent with the clinical finding of myotonia in myotonic dystrophy.     

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIalpha subunit would reveal those tissues and signaling events that require anchored PKA. RIIalpha knockout mice appear normal and healthy. In adult skeletal muscle, RIalpha protein levels increased to partially compensate for the loss of RIIalpha. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIalpha knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA-AKAP interactions. The C subunit colocalized with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIalpha subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIalpha subunit. The potentiation of the L-type Ca2+ channel in RIIalpha knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIalpha is capable of physiologically relevant anchoring interactions.     

Tyrosine phosphorylation of the Kv1.3 potassium channel

Kv1.3, a voltage-dependent potassium channel cloned from mammalian brain and T lymphocytes, contains multiple tyrosine residues that are putative targets for tyrosine kinases. We have examined the tyrosine phosphorylation of Kv1.3, expressed transiently in human embryonic kidney (or HEK) 293 cells, by endogenous and coexpressed tyrosine kinases. Tyrosine phosphorylation is measured by a strategy of immunoprecipitation followed by. Western blot analysis, using antibodies that specifically recognize Kv1.3 and phosphotyrosine. Coexpression of the constitutively active tyrosine kinase v-src, together with Kv1.3, causes a large increase in the tyrosine phosphorylation of the channel protein. This phosphorylation of Kv1.3 can be reversed by treatment with alkaline phosphatase before Western blot analysis. Coexpression with a receptor tyrosine kinase, the human epidermal growth factor receptor, also causes an increase in tyrosine phosphorylation of Kv1.3. The effects of endogenous tyrosine kinases were examined by treating Kv1.3-transfected cells with the specific membrane-permeant tyrosine phosphatase inhibitor pervanadate. Pervanadate treatment causes a time- and concentration-dependent increase in the tyrosine phosphorylation of Kv1.3. This increased tyrosine phosphorylation of Kv1.3 is accompanied by a time-dependent decrease in Kv1.3 current, measured by patch-clamp analysis with cell-attached membrane patches. The pervanadate-induced suppression of current and much of the channel tyrosine phosphorylation are eliminated by mutation of a specific tyrosine residue, at position 449 of Kv1.3, to phenylalanine. Thus, there is a continual phosphorylation and dephosphorylation of Kv1.3 by endogenous kinases and phosphatases, and perturbation of this constitutive phosphorylation/dephosphorylation cycle can profoundly influence channel activity.