Role of KCNQ1 in the cell swelling-induced enhancement of the slowly activating delayed rectifier K(+) current

Cell swelling enhances a slowly activating delayed rectifier K(+) current (I(Ks)) in cardiac cells. This investigation was undertaken to determine which of the two structural units reconstituting the I(Ks) channel, KCNQ1 (KvLQT1) and KCNE1 (minK/IsK), plays a key role in the cell swelling-induced I(Ks) enhancement and to dissect a possible involvement of tyrosine phosphorylation therein. KCNQ1 was transiently expressed alone or together with KCNE1 in a heterologous mammalian cell line. Two distinct whole-cell membrane currents were separately observed during the exposure of transfected cells to various degrees of hyposmotic solutions. A hyposmotic challenge (0.7 times control osmolarity) resulted in about a twofold increase not only in the heteromeric KCNQ1/KCNE1, but also in the homomeric KCNQ1 channel currents. There was no significant difference in the incremental ratio of current amplitude in response to hyposmotic stress between the two KCNQ1-related currents, and the cells expressing the heteromeric channels swelled less than those with the homomeric channels or without the exogenous ones. The cell swelling-induced I(Ks) enhancement was not affected by a protein tyrosine kinase (PTK) inhibitor, by genistein (50 microM), or by an inhibitor of phosphotyrosine phosphatase (PTP), orthovanadate (500 microM), or a nonhydrolyzable ATP analogue, AMP-PNP (5 mM). Taken together, it is very likely that KCNQ1 might primarily participate in the I(Ks) enhancement by osmotic cell swelling. The obligatory dependence of the I(Ks) augmentation on PTK activity remained to be demonstrated, at least, in this expression system.     

Tyrosine kinase inhibitors suppress N-type and T-type Ca2+ channel currents in NG108–15 cells

 Modulation of Ca²⁺ channel activity by protein kinases constitutes one of the major mechanisms regulating neuronal functions. Here, we explored the possible modulation of neuronal Ca²⁺ channels by protein tyrosine kinases (PTKs). To this end, the effects of PTK inhibitors on whole-cell Ba²⁺ currents (I _Ba) through voltage-gated Ca²⁺ channels were analysed in differentiated NG108–15 neuroblastoma × glioma hybrid cells. Genistein suppressed I _Ba in a concentration-dependent fashion (IC₅₀ = 22 μM). Although daidzein, an analogue of genistein that is devoid of PTK inhibitory activity, also suppressed I _Ba, we estimated that specific PTK inhibition by genistein reduced I _Ba amplitude by 30%. In addition, lavendustin A (20 μM) and herbimycin A (20 μM), two other distinct PTK inhibitors, depressed I _Ba by 22% and 20%, respectively. Genistein suppressed N-type and T-type currents, sparing L-type current, and its effect was independent of G protein activation. The results suggest that the activity of neuronal Ca²⁺ channels can be modulated by PTKs, opening the possibility that some of the functions of PTKs in the nervous system are mediated by Ca²⁺ channel modulation. Received: 21 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998     

Molecular basis for genistein-induced inhibition of Kir2.3 currents

Inwardly rectifying potassium channels play an important role in the maintenance of membrane potential in neurons and myocardium. Identification of functional regulation mechanisms concerning these channels may lead to the development of specific modulators for these channels. Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. In this study, we have found that among three members of the Kir family (Kir2.3, Kir2.1, and Kir3.4* [a highly active mutant of Kir3.4, Kir3.4-S143T]) we tested, genistein significantly inhibited Kir2.3 currents. Using the two-electrode voltage clamp technique, we have demonstrated that micromole concentrations of genistein concentration-dependently and reversibly inhibited the currents of Kir2.3 channel expressed in Xenopus oocytes with an IC₅₀ of 16.9 ± 2.8 μM. Using the whole-cell patch-clamp technique, genistein also inhibited the currents of Kir2.3 channel expressed in HEK293 cells with an IC₅₀ of 19.3 ± 3.2 μM. Genistein had little or no effect on Kir2.1 and Kir3.4* currents. The effect of genistein on Kir2.3 currents was not affected by vanadate, a potent protein tyrosine phosphatase inhibitor. Furthermore, the effect of genistein was not mimicked by daidzein, an inactive analogue of genistein, or another potent tyrosine kinase inhibitor, tyrphostin 23. Chimeras between Kir2.3 and Kir2.1 channels were constructed to identify molecular basis that distinguished the effect of genistein on these channels. It was found that the transmembrane domains and the pore region of Kir2.3 channel were important determinant for high sensitivity for genistein inhibition.     

Angiotensin II type 1 receptor mediates partially hyposmotic-induced increase of I Ks current in guinea pig atrium

A repolarizing conduction in the heart augmented by hyposmotic or mechanically induced membrane stretch is the slow component of delayed rectifier K⁺ current (I _Ks). I _Ks upregulation is recognized as a factor promoting appearance of atrial fibrillation (AF) since gain-of-function mutations of the channel genes have been detected in congenital AF. Mechanical stretch activates angiotensin II type 1 (AT₁) receptor in the absence of its physiological ligand angiotensin II. We investigated the functional role of AT₁ receptor in I _Ks enhancement in hyposmotically challenged guinea pig atrial myocytes using the whole-cell patch-clamp method. In atrial myocytes exposed to hyposmotic solution with osmolality decreased to 70% of the physiological level, I _Ks was enhanced by 84.1%, the duration of action potential at 90% repolarization (APD₉₀) was decreased by 16.8%, and resting membrane potential was depolarized (+4.9 mV). The hyposmotic-induced effects on I _Ks and APD₉₀ were significantly attenuated by specific AT₁ receptor antagonist candesartan (1 and 5 μM). Pretreatment of atrial myocytes with protein tyrosine kinase inhibitors tyrphostin A23 and A25 suppressed but the presence of tyrosine phosphatase inhibitor orthovanadate augmented hyposmotic stimulation of I _Ks. The above results implicate AT₁ receptor and tyrosine kinases in the hyposmotic modulation of atrial I _Ks and suggest acute antiarrhythmic properties of AT₁ antagonists in the settings of stretch-related atrial tachyarrhythmias.     

Direct action of genistein on CFTR

 Human cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels were expressed in oocytes from Xenopus laevis after injection of CFTR cRNA and studied with the two-electrode voltage-clamp and the giant patch techniques. The tyrosine kinase inhibitor genistein alone activated a small chloride current in whole oocytes expressing CFTR and substantially increased the chloride current obtained upon stimulation with forskolin and isobutyl methylxanthine (IBMX). In giant excised patches, genistein was unable to open protein-kinase-A-phosphorylated CFTR channels in the absence of ATP, but increased the ATP-induced CFTR channel currents by a factor of 3.8 ± 1.7. This genistein-mediated potentiation in excised patches is independent of protein phosphatase activity, as it is readily reversible, even after complete inhibition of protein kinase A activity. Involvement of protein tyrosine kinases also seems unlikely, because this effect of genistein is not antagonized by high concentrations of the tyrosine phosphatase inhibitor ortho-vanadate. We, therefore, propose a direct interaction of genistein with CFTR, probably at a nucleotide binding site, which leads to a higher open probability. Received: 10 March 1997 / Received after revision and accepted: 22 April 1997     

Regulation of ionic currents by protein kinase A and intracellular calcium in outer hair cells isolated from the guinea-pig cochlea

 Two prominent potassium currents, termed I _K and I _K,n, and a cation current are found in outer hair cells (OHCs) of the guinea-pig cochlea. We report here whole-cell recordings which indicate that the currents are regulated by intracellular factors. 8-bromo-cAMP (500 μM), a membrane-permeable cAMP analogue, activated potassium currents in OHCs in both apical and basal turns of the cochlea. In OHCs from the cochlear apex, the drug effect was largest at potentials positive to –40 mV, indicating I _K as the target. In short cells from the cochlear base, both I _K and I _K,n were affected. The effects of 8-bromo-cAMP could be blocked by the presence of 1 μM H-89 (a protein kinase A inhibitor) in the patch pipette solution. Extracellular application of 10 nM okadaic acid, a protein phosphatase inhibitor, also activated both potassium currents. Currents were also modulated by intracellular calcium. I _K was activated in long cells by photorelease of calcium from the caged compound nitr5. Cation current activation required calcium release by photolysis of DM-nitrophen, a compound releasing more calcium. The results show that OHC potassium channels are regulated by background phosphorylation through protein kinase A and dephosphorylation by protein phosphatase. Cellular calcium also activates I _K and the cation channel, but with different sensitivities. Received: 1 September 1998 / Received after revision: 21 October 1998 / Accepted: 22 October 1998     

Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current

The effect of tyrosine protein kinase inhibitors on the swelling-induced chloride current (I _Cl-swelling of dog atrial myocytes was studied using the whole-cell patch-clamp recording technique. Currents were measured during hyperpolarizing voltage ramps with potassium currents blocked by cesium. Osmolarity was varied using mannitol. Exposure to hypotonic solution (249 mosmol/kg) activated I _Cl-swelling. Hypertonic solution ( 363mosmol/kg) was used to shrink swollen cells and turn off I _Cl-swelling. In studies on the acute effect of tyrosine protein kinase inhibitors each cell was swollen three separate times. Control, treatment, and washout I _Cl-swelling were compared. Genistein (50–80 M) prevented reactivation of I _Cl-swelling without affecting cell size. The effect of genistein partially subsided upon washout. The effect of genistein on I _Cl-swelling was not mimicked by 80 M daidzein, a related compound that does not inhibit tyrosine protein kinases. When intracellular adenosine 5-0-(3-thiotriphosphate (ATP[S]) was used, genistein did not prevent the reactivation of I _Cl-swelling. Intracellular ATP[S] did not result in a persistent activation of I _Cl-swelling when cell size was returned to control. Acute exposure to 1 M herbimycin A or 100 M tyrphostin 51 did not prohibit the activation of I _Cl-swelling. A 24-h exposure to 1 M herbimycin A did inhibit I _Cl-swelling. The results provide important clues regarding the activation mechanism for I _Cl-swelling and suggest that a tyrosine protein phosphorylation may be necessary, but not sufficient, for activation of I _Cl-swelling.     

Protein kinase C stimulates swelling-induced chloride current in canine atrial cells

 The whole-cell patch-clamp technique was used to study the effect of protein kinase C (PKC) stimulation and α-adrenergic agonists on the swelling-induced chloride current (I _Cl,swell) in canine atrial cells. I _Cl,swell was activated by positive-pressure inflation. 4β-Phorbol 12, 13-dibutyrate (PDBu) concentration-dependently stimulated I _Cl,swell. PDBu (500 nM) increased the current density of I _Cl,swell from 9.1±1.3 to 24.2±4.8 pA/pF at +20 mV (n=4). This effect developed slowly, reaching a steady-state after more than 5 min of exposure. 4α-Phorbol 12, 13-dibutyrate (4α-PDBu, 500 nM), an inactive analogue of PDBu, did not affect I _Cl,swell. The effect of PDBu was inhibited by bisindolylmaleimide I. After down regulation of PKC by phorbol 12-myristate 13-acetate (PMA, 1.6 μM, 24 h), I _Cl,swell no longer responded to PDBu (n=4). Neither the basal whole-cell current (prior to cell inflation) nor inflation-induced I _Cl,swell were affected by PKC down regulation. Phenylephrine did not affect I _Cl,swell. We conclude that PKC activity stimulates and does not prevent the activation of dog atrial I _Cl,swell. These results contrast with reports of PKC-dependent inhibition of rabbit atrial I _Cl,swell and currents conducted by ClC-3, a putative clone for I _Cl,swell. The data suggest species-dependent variations in the modulation of cardiac I _Cl,swell by PKC. Received: 19 June 1998 / Received after revision: 15 September 1998 / Accepted: 25 September 1998     

Urocortin hyperpolarizes stomach smooth muscle via activation of Ca2+-sensitive K+ currents

The effect of urocortin (Uro), a recently discovered neuropeptide with selectivity towards corticotropin-releasing hormone type 2 receptor, was tested on whole cell currents expressed by guinea-pig gastric antrum smooth muscle cells. Uro (1 pmol/l – 1 nmol/l) caused a concentration-dependent increase of Ca²⁺-sensitive K currents (I _K) up to 500% as compared to control currents and did not affect the kinetics and voltage-dependence of inward Ca²⁺ currents. The I _K-increasing effect of Uro was fully antagonized by preliminary emptying of intracellular Ca²⁺ stores with ryanodine and cyclopiazonic acid, as well as by bath application of selective blockers of adenylyl cyclase and cAMP-dependent protein kinase (PKA), but not by inhibitors of guanylyl cyclase, cGMP-dependent protein kinase, and protein kinase C. Comparable I _K increase was obtained by forskolin (activator of adenylyl cyclase), Sp-cAMPS (activator of PKA), or by intracellular application of the catalytic subunit of PKA. It was concluded that Uro binds to a selective receptor in antral smooth muscle cells where it stimulates I _K via PKA-dependent increase of Ca²⁺ concentration near the plasma membrane due to enhanced release from intracellular calcium stores.     

Corticotropin-releasing hormone acts on guinea pig ileal smooth muscle via protein kinase A

In contraction studies corticotropin-releasing hormone (CRH) was found to relax ileal but not gastric and jejunal smooth muscles of the guinea-pig, precontracted with BaCl₂. Under whole-cell patch-clamp conditions, CRH concentration-dependently activated Ca²⁺-sensitive K⁺ currents (I _K) with ED₅₀=20 pM at 100 nM and ED₅₀=0.13 pM at 500 nM intracellular Ca²⁺ respectively. This increase was accompanied by significant hyperpolarization of the cell membranes. CRH 9–41 peptide fragment did not affect I _K amplitude, membrane potential or contraction. The CRH-induced increase of I _K densities was accelerated in the presence of high intracellular Ca²⁺ concentrations (500 nM) and was abolished by pretreatment of cells with either ryanodine or thapsigargin, which cause depletion of intracellular Ca²⁺ stores, as well as in cells treated under conditions prohibiting intracellular Ca²⁺ store refilling. The effect of CRH on I _K was not affected by bath application of various selective inhibitors of membrane-bound phospholipases, protein kinase C, cGMP-dependent protein kinase or Ca²⁺/calmodulin-dependent protein kinase II, but was effectively antagonized by blockers of protein kinase A (PKA) or adenylyl cyclase. Neither forskolin nor the catalytic subunit of PKA could mimic the effect of CRH on I _K. Thus, it was suggested that CRH exerts its relaxing activity on ileal smooth muscle cells via PKA-dependent phosphorylation of some intracellular target coupled to sarcoplasmic reticulum Ca²⁺ storage machinery. Received: 18 January 1999 / Received after revision: 8 March 1999 / Accepted: 9 March 1999     

Action potentials,ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture

 Adult rabbit ventricular myocytes were cultured in a basic medium (Medium 199) for up to 6 days to assess preservation of morphology and ion channel currents. In culture, cells remained rod shaped and striated but their ends became progressively rounded. Cell cross-sectional area declined slightly (by 14%) over the first 24 h, in contrast, whole-cell capacitance (which reflects external surface membrane plus membrane infoldings) decreased by 42% over the same time. Using whole-cell patch-clamp, we observed that the typical ”N” shape steady-state current-voltage (I-V) relation became flattened after 24 h in culture. L-type Ca channel density was assessed as barium flux (I _Ba,L) via the channel. I _Ba,L (normalised to cell capacitance) declined by 50% after 24 h and recovered partially by days 4 and 6. The density of inward rectifier K current declined by 54% after 24 h and showed no recovery subsequently. In contrast, there was no significant decline in the density of transient outward K current after 24 h, but it declined subsequently by 65% after 6 days. We speculate that the time course of change in each ion channel density may reflect a change in pattern of ion channel expression, or differential membrane loss since the density of transverse tubules decreased by 57% after 6 days in culture. These results suggest that even by 24 h in culture, ion channel density in myocytes has changed substantially from the acutely isolated state. Received: 24 July 1995 / Received after revision: 15 November 1995 / Accepted: 15 November 1995     

Involvement of tyrosine kinase in the hyposmotic stimulation of I Ks in guinea-pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the hyposmotic stimulation of cardiac I _Ks, a slowly activating delayed-rectifier K⁺ current that promotes repolarization of the action potential. The current was recorded from whole-cell-configured guinea-pig ventricular myocytes before, during, and after their exposure to solution whose osmolarity was 0.75 times normal. Exposure to hyposmotic solution caused a near-doubling of the amplitude of I _Ks, with little change in the voltage dependence of current activation. Stable, hyposmotically stimulated I _Ks (I _Ks,Hypo) was decreased by broadspectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC₅₀ ≈ 5 μM) and tyrphostin A25 (IC₅₀ 15.8 ± 1.6 μM) but not by TK-inactive tyrphostin analogs, suggesting that tyrosine phosphorylation is important for maintenance of the current. In agreement with that view, we found that the TK-inhibitor action on I _Ks,Hypo was strongly antagonized by vanadate compounds known to inhibit phosphotyrosyl phosphatase. When myocytes were pretreated with TK inhibitors, the stimulation of I _Ks was attenuated in a concentration-dependent manner. The attenuation was not due to concomitant attenuation of a stimulation of tyrosine phosphorylation because neither the stimulation of I _Ks nor its rate of decay following removal of hyposmotic solution was affected by pretreatment with vanadates. We suggest that the stimulation of I _Ks by hyposmotic solution is dependent on a basal tyrosine phosphorylation that modulates a swelling-induced I _Ks-stimulatory signal and/or the receptivity of Ks channels to that signal.     

Roles of the voltage-gated K+ channel subunits, Kv 1.5 and Kv 1.4, in the developmental changes of K+ currents in cultured neonatal rat ventricular cells

 To investigate the roles of voltage-gated K⁺ channel subunits, Kv 1.5 and Kv 1.4, in the developmental regulation of K⁺ currents, we determined the K⁺ channel activities and the distributions of K⁺ channel subunits in the same single cultured neonatal rat ventricular cells, using a whole-cell patch-clamp technique and an immunocytochemical analysis of K⁺ channel proteins. In 5-day cultured cells, two types of 4-aminopyridine (4-AP)-sensitive and rapidly activating K⁺ currents, the transient outward current (I_to) and the ultrarapid delayed rectifier (I_Kur), could be distinguished. A small proportion of 5-day cells expressing sole I_Kur demonstrated an intense anti-Kv 1.5 antibody labeling with punctate distribution outlining the cells, while a weak staining was observed in the majority of 5-day cells expressing sole I_to. At day 15 of cell culture, only I_to was present with a lower level of the immunocytochemical expression of Kv 1.5 channel protein. Staining of the Kv 1.4 channel protein was qualitatively similar in the 5-day cells expressing either I_to or I_Kur. However, anti-Kv 1.4 antibody did not label the 15-day cultured cells showing remarkably increased I_to density. Our results strongly indicate that the Kv 1.5 channel expression may underlie the developmental regulation of I_Kur, while Kv 1.4 channel does not contribute to the postnatal increase in I_to. Received: 19 December 1996 / Received after revision: 9 February 1997 / Accepted: 19 February 1997     

Cell swelling causes the action potential duration to shorten in guinea-pig ventricular myocytes by activating I KATP

 In guinea-pig ventricular myocytes, cell swelling by incubation in hypotonic solution caused a pronounced shortening of the action potential duration (APD₉₀: 15.5±14.6% compared to control; mean ± SD) after a latency of 12 min when the intracellular ATP concentration was 2 mM. This shortening was partially reversible within 10 min after reperfusion with isotonic solution (APD₉₀: 80.5±12.1% compared to control). With 5 mM intracellular ATP in the pipette electrode, the effect of cell swelling on the action potential was significantly reduced. Incubation with 1 μM glibenclamide, a blocker of the ATP-dependent K⁺ current (I _KATP), abolished the swelling-induced shortening of the action potential duration, whereas incubation with 0.5 mM 4,4’-diisothiocyanatostilbene-2,2’-disulphonic acid (DIDS), a blocker of the swelling-induced Cl^– current (I _Cl,swell), had no effect on the action potential duration in hypotonic solution. Simultaneous measurements of membrane currents substantiate that I _KATP is the current that underlies this effect. These results suggest that in the ischaemic myocardium I _KATP may be partially activated by cell swelling, resulting in a shortening of the action potential duration before the intracellular ATP concentration has fallen below 2 mM. Received: 30 March 1998 Received after revision: 7 July 1998 Accepted: 25 July 1998     

The sustained inward current in sino-atrial node cells of guinea-pig heart

 Single myocytes were dissociated from the sino-atrial (SA) node of guinea-pig hearts. Only a quite small fraction of the cell population showed spontaneous action potentials and these cells were characterized by the presence of the hyperpolarization-activated cation current I _f , the delayed rectifier K⁺ current I _K and the L-type Ca²⁺ current I _Ca,L as well as by the absence of both the transient outward current I _to and the inward rectifier K⁺ current I _K,1. After blocking I _f and I _K, depolarizing pulses from –80 mV revealed a large nicardipine-sensitive late current (NSLC). The NSLC was scarcely affected by decreasing extracellular [Ca²⁺] ([Ca²⁺]_o) from 1.8 to 0.1 mM, while it was decreased significantly by depleting [Na⁺]_o, differently from I _Ca,L. NSLC was blocked by nicardipine and was increased by Bay K 8644. NSLC was increased by isoprenaline and the additional application of acetylcholine reversed the increase of this current. We conclude that NSLC is largely composed of I _st described in the rabbit SA node pacemaker cells, and that I _st is unique for the pacemaker cells in mammalian SA node cells. Most of the quiescent cells showed neither I _f nor I _st. Received: 22 July 1996 / Received after revision: 30 September 1996 / Accepted: 9 October 1996     

Analysis of the time course of calcium-activated chloride “tail” currents in rabbit portal vein smooth muscle cells

 The time course of calcium-activated chloride ”tail” currents (I _tail) in single cells of the rabbit portal vein was studied. These currents were activated by the influx of calcium through voltage-dependent calcium channels (VDCCs). At –50 mV, I _tail decayed exponentially with a time constant (τ) of 80–100 ms that was independent of amplitude and was similar to the τof the decay of spontaneous transient inward currents (STICs; calcium-activated chloride currents). The decays of the STIC and I _tail had a similar voltage dependence between –50 and –110 mV and were similarly affected by the chloride channel blocker, niflumic acid. However, at more positive potentials (–20 to +40 mV), I _tail was sustained for the duration of the test pulse in most cells, in contrast to STICs which decayed exponentially. At very positive potentials (e.g. +100 mV), when little calcium enters the cell through VDCCs, I _tail decayed exponentially. Measurement of calcium current (I _Ca) at various potentials showed that the VDCCs did not inactivate fully at potentials between –20 and +30 mV. We propose that at negative potentials the decay of I _tail is determined by slow gating of the chloride channel, but at positive potentials a sustained I _tail is produced by persistent influx of calcium through non-inactivating VDCCs. Received: 19 March 1996 / Received after revision: 22 May 1996 / Accepted: 23 May 1996     

Calcium-dependent changes in potassium currents in guinea-pig coronary artery smooth muscle cells after acute cobalt loading in vivo

The aim of the present study was to determine whether cobalt poisoning induces haem oxidase isoenzyme-1 (HO-1) in coronary artery smooth muscle, or accounts for any changes in coronary smooth muscle cell (SMCs) membrane ionic currents that could result from this type of heavy metal poisoning. In SMCs isolated from cobalt-treated guinea-pig coronaries, K⁺ channel currents (I _K) were much smaller than those in cells isolated from non-treated animals. Haemin (HO substrate) increased I _K concentration dependently. This effect was mimicked by 1% CO and was abolished by pretreatment of cells with a competitive HO inhibitor, by inhibitors of guanylyl cyclase, protein kinase G or phospholipase C, as well as by blocking inositol trisphosphate-dependent Ca release, or sarcoplasmic reticulum Ca-ATPase, or by bathing cells in Ca-free external solution. Expression of the Na/Ca exchanger-1 (NCX-1) protein was reduced substantially in SMCs from coronary arteries of cobalt-treated animals. No expression of HO-1 was detected. It is concluded that acute cobalt poisoning in vivo depresses Ca-sensitive K currents via CO-dependent modulation of intracellular calcium availability, most probably by suppressing the expression of NCX-1 protein.     

Protein tyrosine phosphorylation and the regulation of KCl cotransport in trout erythrocytes

Electroneutral salt transporters are activated and deactivated by changes to the phosphorylation status either of the transporter itself or of other, as yet unidentified, regulatory proteins. We have studied the effects of an inhibitor of protein tyrosine kinase (PTK), genistein, upon KCl cotransport in trout erythrocytes. We show that Cl-dependent K fluxes activated by physiological stimuli, i.e. oxygenation and β-adrenergic agonists, are rapidly and completely blocked by genistein, whilst the inactive analogue of genistein, daidzein, had no effect. By contrast, the protein tyrosine phosphatase (PTP) inhibitor, vanadate (V), caused a slow but strong activation of an inactive cotransporter. This vanadate (V) activated flux was inhibited by genistein as well as by the serine/threonine phosphatase (PSP) inhibitor, calyculin A. However, genistein had no effect upon the activation of the cotransporter by the protein (serine/threonine) kinase (PSK) inhibitor, staurosporine, or by N-ethylmaleimide, which also appears to act by inhibiting a PSK. These results are consistent with a sequential scheme of at least two tyrosine phosphorylation events which lie upstream to the serine/threonine phosphorylation sites in the signal transduction pathway leading from stimulus to transporter activation. The regulation of the activity of KCl cotransporter appears to involve a complex series of phosphorylation reactions. Received: 4 March 1996/Accepted: 1 May 1996     

Protein tyrosine kinase activation provides an early and obligatory signal in anti-FRP-1/CD98/4F2 monoclonal antibody induced cell fusion mediated by HIV gp160

The mechanism by which anti-fusion regulatory protein-1 (FRP-1) monoclonal antibody (mAb) induced cell fusion was investigated using U2ME-7 cells that are CD4⁺U937 cells transfected with the HIV gp160 gene. Protein kinase inhibitors (H-7, H-89, herbimycin A and genistein) suppressed cell fusion of Cd⁺U2ME-7 cells induced by anti-FRP-1 mAb. H-7 and H-89 also inhibited the cell aggregation, but herbimycin A and genistein did not. Intriguingly, only when herbimycin A was added either before or simultaneously with addition of anti-FRP-1 mAb, was cell fusion suppressed, suggesting that tyrosine kinase is related with the initial step of polykaryocyte formation. Anti-FRP-1 mAb induced the rapid tyrosine phosphorylation of multiple cellular proteins. These effects occurred within 1 min and returned to near baseline by 60 min. The rapid tyrosine phosphorylation was suppressed by herbimycin A and genistein. Although it remains to be determined which protein tyrosine kinase(s) is involved in this response, pp130 tyrosine phosphorylation appears to be a specific and early signal transmitted after the interaction of FRP-1 with a specific antibody. pp130 was present in the cytosol fraction and was distinct from pp125^FAK, p130^CAS, vinculin, and β1-integrin. Thus, our study may present evidence for a novel pathway of protein tyrosine kinases that phosphorylate specific, still unknown protein substrates during polykaryocyte formation. Received: 30 May 1997     

α1-Adrenoceptor agonists and IGF-1, myocardial hypertrophic factors, regulate the Kv1.5 K+ channel expression differentially in cultured newborn rat ventricular cells

 Interest has arisen concerning the importance of α-adrenergic function and insulin-like growth factor-1 (IGF-1) in cardiac remodelling. The hypothesis that these two factors may underlie the regulation of voltage-gated K⁺ channel expression in hypertrophied cardiomyocytes was tested by performing Western blot analysis of the Kv1.5 K⁺ channel α-subunit in cultured newborn rat ventricular cells. Myocyte size was quantified by surface area and total cell protein concentration. Cell exposure to the α₁-adrenoceptor agonist phenylephrine (PE, 20 μM) and IGF-1 (60 ng/ml) for 72 h both induced a significant increase of cell size indicating myocyte hypertrophy, which could be separately blocked by the protein kinase C inhibitor staurosporine (20 nM) and the tyrosine kinase inhibitor genistein (15 μM). Western blots of cell proteins prepared from myocyte cultures showed a single protein band at 75 kD recognized by the anti-Kv1.5 antibody, and demonstrated a 56% reduction in the Kv1.5 immunoreactive protein level in the PE-treated cell preparations. This suppression was not affected by staurosporine, but was remarkably attenuated by W7 (20 μM), a selective calmodulin antagonist. In contrast to PE, a 48% enhancement of the protein expression of Kv1.5 channel was induced by IGF-1 and this stimulation was specifically blocked by genistein. Our findings suggest that the differential regulation of cardiac Kv1.5 K⁺ channel expression can be produced by α₁-adrenoceptor activation and IGF-1 via distinctive signalling pathways. Calmodulin-dependent kinase and tyrosine kinase contribute importantly to the α₁-adrenoceptor-mediated decrease and the IGF-1-mediated increase in cardiac Kv1.5 K⁺ channel expression, respectively. Received: 19 August 1997 / Accepted: 14 January 1998