Palmitoylation and membrane association of the stress axis regulated insert (STREX) controls BK channel regulation by protein kinase C.

Large-conductance, calcium- and voltage-gated potassium (BK) channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis regulated exon (STREX) at splice site 2 inverts BK channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal channel activity by PKC in the insertless (ZERO) splice variant, STREX channels were completely resistant to PKC. STREX channel mutants in which Ser(700), located between the two regulatory domains of K(+) conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ∼50% decrease in maximal channel activity, whereas the S700A mutant retained its normal activity. BK channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK channels where PKC fails to phosphorylate and to inhibit channel activity. Importantly, PKA which inhibits channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion channel palmitoylation and phosphorylation in regulation of cellular excitability.

Inhibition of HERG1 K+ channel protein expression decreases cell proliferation of human small cell lung cancer cells.

HERG (human ether-à-go-go-related gene) K(+) currents fulfill important ionic functions in cardiac and other excitable cells. In addition, HERG channels influence cell growth and migration in various types of tumor cells. The mechanisms underlying these functions are still not resolved. Here, we investigated the role of HERG channels for cell growth in a cell line (SW2) derived from small cell lung cancer (SCLC), a malignant variant of lung cancer. The two HERG1 isoforms (HERG1a, HERG1b) as well as HERG2 and HERG3 are expressed in SW2 cells. Inhibition of HERG currents by acute or sustained application of E-4031, a specific ERG channel blocker, depolarized SW2 cells by 10-15 mV. This result indicated that HERG K(+) conductance contributes considerably to the maintenance of the resting potential of about -45 mV. Blockage of HERG channels by E-4031 for up to 72 h did not affect cell proliferation. In contrast, siRNA-induced inhibition of HERG1 protein expression decreased cell proliferation by about 50%. Reduction of HERG1 protein expression was confirmed by Western blots. HERG current was almost absent in SW2 cells transfected with siRNA against HERG1. Qualitatively similar results were obtained in three other SCLC cell lines (OH1, OH3, H82), suggesting that the HERG1 channel protein is involved in SCLC cell growth, whereas the ion-conducting function of HERG1 seems not to be important for cell growth.

Expression pattern of Kv11 (Ether à-go-go-related gene; erg) K+ channels in the mouse retina.

In response to light, most retinal neurons exhibit gradual changes in membrane potential. Therefore K+ channels that mediate threshold currents are well-suited for the fine-tuning of signal transduction. In the present study we demonstrate the expression of the different Kv11 (ether-à-go-go related gene; erg) channel subunits in the human and mouse retina by RT PCR and quantitative PCR, respectively. Immunofluorescence analysis with cryosections of mouse retinae revealed the following local distribution of the three Kv11 subunits: Kv11.1 (m-erg1) displayed the most abundant expression with the strongest immunoreactivity in rod bipolar cells. In addition, immunoreactivity was found in the inner part of the outer plexiform layer (OPL), in the inner plexiform layer (IPL) and in the inner segments of photoreceptors. Immunoreactivity for Kv11.2 (m-erg2) was observed in the outer part of the OPL and throughout the IPL. Double-labeling for vGluT1 or synaptophysin indicated a mainly presynaptic localization of Kv11.2. While no significant staining for Kv11.3 (m-erg3) was detected in the neuronal retina, strong Kv11.3 immunoreactivity was present in the apical membrane of the retinal pigment epithelium. The different expression levels were confirmed by real-time PCR showing almost equal levels of Kv11.1 and Kv11.2, while Kv11.3 mRNA expression was significantly lower. The two main splice variants of Kv11.1, isoforms a and b were detected in comparable levels suggesting a possible formation of cGMP/cGK-sensitive Kv11.1 channels in photoreceptors and rod bipolar cells. Taken together, the immunohistological results revealed different expression patterns of the three Kv11 channels in the mouse retina supposing distinct physiological roles.

Dual role of protein kinase C on BK channel regulation.

Large conductance voltage- and Ca(2+)-activated potassium channels (BK channels) are important feedback regulators in excitable cells and are potently regulated by protein kinases. The present study reveals a dual role of protein kinase C (PKC) on BK channel regulation. Phosphorylation of S(695) by PKC, located between the two regulators of K(+) conductance (RCK1/2) domains, inhibits BK channel open-state probability. This PKC-dependent inhibition depends on a preceding phosphorylation of S(1151) in the C terminus of the channel alpha-subunit. Phosphorylation of only one alpha-subunit at S(1151) and S(695) within the tetrameric pore is sufficient to inhibit BK channel activity. We further detected that protein phosphatase 1 is associated with the channel, constantly counteracting phosphorylation of S(695). PKC phosphorylation at S(1151) also influences stimulation of BK channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S(1151)A mutant channel is activated by PKA only, the phosphorylation of S(1151) by PKC renders the channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S(695) by PKC or introducing a phosphomimetic aspartate at this position (S(695)D) renders BK channels insensitive to the stimulatory effect of PKG or PKA. Therefore, our findings suggest a very dynamic regulation of the channel by the local PKC activity. It is shown that this complex regulation is not only effective in recombinant channels but also in native BK channels from tracheal smooth muscle.

Modulation of cardiac ERG1 K(+) channels by cGMP signaling.

Different K(+) currents have been implicated in the myocardial action potential repolarization including the I(Kr). ERG1 alpha subunits, identified as the molecular correlate of I(Kr), have been shown to form heteromultimeric channels in the heart and their activity is modulated by a complex interplay of signal transduction events. Using electrophysiological techniques, we examined the effects of the cGMP-analogue 8-Br-cGMP on rat and guinea-pig papillary action potential duration (APD), on the biophysical properties of heterologously expressed homo- and heteromeric ERG1 channels, and on cardiac I(Kr). 8-Br-cGMP prolonged APD by about 25% after pharmacological inhibition of L-type Ca(2+) currents and I(Ks). The prolongation was completely abolished by prior application of the hERG channel blocker E-4031 or the protein kinase G (PKG) inhibitor Rp-8-Br-cGMPS. Expression analysis revealed the presence of both ERG1a and -1b subunits in rat papillary muscle. Both 8-Br-cGMP and ANP inhibited heterologously expressed ERG1b and even stronger ERG1a/1b channels, whereas ERG1a channels remained unaffected. The inhibitory 8-Br-cGMP effects were PKG-dependent and involved a profound ERG current reduction, which was also observed with cardiac AP clamp recordings. Measurements of I(Kr) from isolated mouse cardiomyocytes using Cs(+) as charge carrier exhibited faster deactivation kinetics in atrial than in ventricular myocytes consistent with a higher relative expression of ERG1b transcripts in atria than in ventricles. 8-Br-cGMP significantly reduced I(Kr) in atrial, but not in ventricular myocytes. These findings provide first evidence that through heteromeric assembly ERG1 channels become a critical target of cGMP-PKG signaling linking cGMP accumulation to cardiac I(Kr) modulation.

Erg K+ currents modulate excitability in mouse mitral/tufted neurons.

Different erg (ether-à-go-go-related gene; Kv11) K+ channel subunits are expressed throughout the brain. Especially mitral cells of the olfactory bulb are stained intensely by erg1a, erg1b, erg2, and erg3 antibodies. This led us to study the erg current in mitral/tufted (M/T) neurons from mouse olfactory bulb in primary culture. M/T neurons were identified by their morphology and presence of mGluR1 receptors, and RT-PCR demonstrated the expression of all erg subunits in cultured M/T neurons. Using an elevated external K+ concentration, a relatively uniform erg current was recorded in the majority of M/T cells and isolated with the erg channel blocker E-4031. With 4-s depolarizations, the erg current started to activate at -65 mV and exhibited half maximal activation at -51 mV. An increase in the external K+ concentration resulted in an increase in erg whole-cell conductance. The specific group 1 mGluR agonist, DHPG, which depolarizes mitral cells, reduced erg channel availability. DHPG accelerated erg current deactivation, reduced the maximum current amplitude, and shifted availability and activation curves to more depolarized potentials. A pharmacological block of erg channels depolarized the resting potential of M/T cells and clearly demonstrated the involvement of erg channels in the control of mitral cell excitability.

M2 muscarinic receptors induce airway smooth muscle activation via a dual, Gbetagamma-mediated inhibition of large conductance Ca2+-activated K+ channel activity.

Airway smooth muscle is richly endowed with muscarinic receptors of the M(2) and M(3) subtype. Stimulation of these receptors inhibits large conductance calcium-activated K(+) (BK) channels, a negative feed back regulator, in a pertussis toxin-sensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M(2) or M(3) receptor (M(2)R or M(3)R). In M(2)R- but not M(3)R-expressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M(2)R-induced inhibition was abolished by pertussis toxin treatment or overexpression of the Gbetagamma scavenger transducin-alpha. In inside-out patches, direct application of 300 nm purified Gbetagamma decreased channel open probability by 55%. The physical interaction of Gbetagamma with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of phospholipase C as well as protein kinase C activities also reversed the CCh effect but to a smaller (approximately 20%) extent. Mouse tracheal cells responded similarly to CCh, purified Gbetagamma and phospholipase C/protein kinase C inhibition as M(2)R-expressing HEK293 cells. Our results demonstrate that airway M(2)Rs inhibit BK channels by a dual, Gbetagamma-mediated mechanism, a direct membrane-delimited interaction, and the activation of the phospholipase C/protein kinase C pathway.

Erg K+ channels modulate contractile activity in the bovine epididymal duct.

The expression and functional role of ether-à-go-go-related gene (erg) K+ channels were examined in the bovine epididymal duct. Sperm transit through the epididymal duct relies on spontaneous phasic contractions (SC) of the peritubular smooth muscle wall. Isometric tension studies revealed SC-enhancing effects of the erg channel blockers E-4031, dofetilide, cisapride, and haloperidol and SC-suppressing effects of the activator NS-1643. In the corpus epididymidis, EC50 values of 32 nM and 8.3 microM were determined for E-4031 and NS-1643, respectively. E-4031 was also able to elicit contraction in epithelium-denuded corpus segments, which lacked SC. In the cauda region, E-4031 and NS-1643 exerted effects on agonist-induced contraction similar to those observed in the proximal duct. Experiments with nifedipine and thapsigargin suggested that the excitatory effects of E-4031 depended mainly on external calcium influx and not on intracellular calcium release. Western blot and RT-PCR assays revealed the expression of both, erg1a and erg1b, in all duct regions. Because erg1b appears to predominate in the epididymal duct, patch-clamp experiments were performed on heterologously expressed erg1b channels to investigate the sensitivity of this splice variant to NS-1643. In contrast to its effects on erg1a, NS-1643 induced a concentration-dependent current increase mainly due to a marked leftward shift in erg1b channel activation by approximately 30 mV at 10 microM, explaining the inhibitory effect of the drug on epididymal SC. In summary, these data provide strong evidence for a physiological role of erg1 channels in regulating epididymal motility patterns.

Differential modulation of bovine epididymal activity by oxytocin and noradrenaline.

Passage of spermatozoa through the epididymis and emission of sperm during ejaculation are based on spontaneous and induced contractions of epididymal peritubular muscle layers. This study deals with the ejaculation-relevant factors noradrenaline (NA) and oxytocin (OT) and their contractile effects in the course of the bovine epididymal duct. Muscle tension recording revealed excitatory effects of NA in all duct regions. A peculiarity was found in a duct section between the mid-cauda and ductus deferens, where the responsiveness to NA was particularly faint in comparison with the adjacent regions. NA-induced contraction was primarily mediated by postjunctional alpha(2)-adrenoceptors (ADRA) in the caput and corpus regions, and by alpha(1)-ADRA in the cauda region. Contrary to NA, OT exerted regionally varying effects. The peptide induced contraction in intact and epithelium-denuded caput as well as in epithelium-denuded corpus segments but had a relaxant net effect in intact corpus and proximal cauda segments. Within the mid-cauda, OT evoked strong contraction, which progressively decreased distally. Receptor specificity of the epididymal OT effects was verified using the selective OT receptor (OTR) agonist [Thr(4),Gly(7)]OT and vasopressin. OTR immunoreactivity was detected in the epididymal peritubular muscle wall and epithelial principal cells. RT-PCR analysis confirmed the presence of OTR in all duct regions. In summary, different contractile responses to OT and NA occur in the course of the epididymal duct, possibly preventing excessive sperm transport through the corpus and serving orthograde emission of sperm during ejaculation.

Effects of TRH on heteromeric rat erg1a/1b K+ channels are dominated by the rerg1b subunit.

The erg1a (HERG) K+ channel subunit and its N-terminal splice variant erg1b are coexpressed in several tissues and both isoforms have been shown to form heteromultimeric erg channels in heart and brain. The reduction of erg1a current by thyrotropin-releasing hormone (TRH) is well studied, but no comparable data exist for erg1b. Since TRH and TRH receptors are widely expressed in the brain, we have now studied the different TRH effects on the biophysical properties of homomeric rat erg1b as well as heteromeric rat erg1a/1b channels. The erg channels were overexpressed in the clonal somatomammotroph pituitary cell line GH3/B6, which contains TRH receptors and endogenous erg channels. Compared to rerg1a, homomeric rerg1b channels exhibited not only faster deactivation kinetics, but also considerably less steady-state inactivation, and half-maximal activation occurred at about 10 mV more positive potentials. Coexpression of both isoforms resulted in erg currents with intermediate properties concerning the deactivation kinetics, whereas rerg1a dominated the voltage dependence of activation and rerg1b strongly influenced steady-state inactivation. Application of TRH induced a reduction of maximal erg conductance for all tested erg1 currents without effects on the voltage dependence of steady-state inactivation. Nevertheless, homomeric rerg1b channels significantly differed in their response to TRH from rerg1a channels. The TRH-induced shift in the activation curve to more positive potentials, the dramatic slowing of activation and the acceleration of deactivation typical for rerg1a modulation were absent in rerg1b channels. Surprisingly, most effects of TRH on heteromeric rerg1 channels were dominated by the rerg1b subunit.

Fast erg K+ currents in rat embryonic serotonergic neurones.

Ether-á-go-go-related gene (erg) channels form one subfamily of the ether-á-go-go (EAG) K(+) channels and all three erg channels (erg1-3) are expressed in the brain. In the present study we characterize a fast erg current in neurones in primary culture derived from the median part of rat embryonic rhombencephala (E15-16). The relatively uniform erg current was regularly found in large multipolar serotonergic neurones, and occurred also in other less well characterized neurones. The erg current was blocked by the antiarrhythmic substance E-4031. Single-cell RT-PCR revealed the expression of erg1a, erg1b, erg2 and erg3 mRNA in different combinations in large multipolar neurones. These cells also contained neuronal tryptophan hydroxylase, a key enzyme for serotonin production. To characterize the molecular properties of the channels mediating the native erg current, we compared the voltage and time dependence of activation and deactivation of the neuronal erg current to erg1a, erg1b, erg2 and erg3 currents heterologously expressed in CHO cells. The biophysical properties of the neuronal erg current were well within the range displayed by the different heterologously expressed erg currents. Activation and deactivation kinetics of the neuronal erg current were fast and resembled those of erg3 currents. Our data suggest that the erg channels in rat embryonic rhombencephalon neurones are heteromultimers formed by different erg channel subunits.

Nitric oxide donor sodium nitroprusside dilates rat small arteries by activation of inward rectifier potassium channels.

The role of vascular smooth muscle inward rectifier K+ (K(IR)) channels in the mechanisms underlying vasodilation is still unclear. The hypothesis that K(IR) channels are involved in sodium nitroprusside (SNP)-induced dilation of rat-tail small arteries was tested. SNP relaxed tail small arteries with an EC50 of 2.6x10(-8) mol/L. Endothelium removal did not attenuate this effect. Vessel pretreatment with hydroxocobalamin, a nitric oxide (NO) scavenger, but not with rhodanese and sodium thiosulfate, inactivators of cyanide (CN), abolished the SNP effect. Vessel pretreatment with 10(-5) mol/L Ba2+, a specific blocker of K(IR) channels at micromolar concentrations, reduced the SNP effect. Low concentrations of K+ dilated the vessels; this effect was attenuated largely after pretreatment with 3x10(-5) mol/L Ba2+. In freshly isolated smooth muscle cells, a barium-sensitive current was observed at potentials negative to the potassium equilibrium potential. Application of 10(-4) mol/L SNP increased the barium-sensitive current 1.79+/-0.23-fold at -100 mV and hyperpolarized the membrane potential by 8.6+/-0.5 mV. In tissue from freshly dissected vessels, transcripts for K(IR) 2.1 and 2.2, but not for K(IR) 2.3 and 2.4, were found. However, only K(IR) 2.1 antibodies immunostained the tunica media of the vessel. These data suggest that vascular smooth muscle K(IR) 2.1 channels are involved in the SNP-induced dilation of rat-tail small arteries.