Clinically Relevant KCNQ1 Variants Causing KCNQ1-KCNE2 Gain-of-Function Affect the Ca2+ Sensitivity of the Channel.

Dominant KCNQ1 variants are well-known for underlying cardiac arrhythmia syndromes. The two heterozygous KCNQ1 missense variants, R116L and P369L, cause an allelic disorder characterized by pituitary hormone deficiency and maternally inherited gingival fibromatosis. Increased K+ conductance upon co-expression of KCNQ1 mutant channels with the beta subunit KCNE2 is suggested to underlie the phenotype; however, the reason for KCNQ1-KCNE2 (Q1E2) channel gain-of-function is unknown. We aimed to discover the genetic defect in a single individual and three family members with gingival overgrowth and identified the KCNQ1 variants P369L and V185M, respectively. Patch-clamp experiments demonstrated increased constitutive K+ conductance of V185M-Q1E2 channels, confirming the pathogenicity of the novel variant. To gain insight into the pathomechanism, we examined all three disease-causing KCNQ1 mutants. Manipulation of the intracellular Ca2+ concentration prior to and during whole-cell recordings identified an impaired Ca2+ sensitivity of the mutant KCNQ1 channels. With low Ca2+, wild-type KCNQ1 currents were efficiently reduced and exhibited a pre-pulse-dependent cross-over of current traces and a high-voltage-activated component. These features were absent in mutant KCNQ1 channels and in wild-type channels co-expressed with calmodulin and exposed to high intracellular Ca2+. Moreover, co-expression of calmodulin with wild-type Q1E2 channels and loading the cells with high Ca2+ drastically increased Q1E2 current amplitudes, suggesting that KCNE2 normally limits the resting Q1E2 conductance by an increased demand for calcified calmodulin to achieve effective channel opening. Our data link impaired Ca2+ sensitivity of the KCNQ1 mutants R116L, V185M and P369L to Q1E2 gain-of-function that is associated with a particular KCNQ1 channelopathy.

Gain-of-Function Mutations in KCNN3 Encoding the Small-Conductance Ca2+-Activated K+ Channel SK3 Cause Zimmermann-Laband Syndrome.

Zimmermann-Laband syndrome (ZLS) is characterized by coarse facial features with gingival enlargement, intellectual disability (ID), hypertrichosis, and hypoplasia or aplasia of nails and terminal phalanges. De novo missense mutations in KCNH1 and KCNK4, encoding K+ channels, have been identified in subjects with ZLS and ZLS-like phenotype, respectively. We report de novo missense variants in KCNN3 in three individuals with typical clinical features of ZLS. KCNN3 (SK3/KCa2.3) constitutes one of three members of the small-conductance Ca2+-activated K+ (SK) channels that are part of a multiprotein complex consisting of the pore-forming channel subunits, the constitutively bound Ca2+ sensor calmodulin, protein kinase CK2, and protein phosphatase 2A. CK2 modulates Ca2+ sensitivity of the channels by phosphorylating SK-bound calmodulin. Patch-clamp whole-cell recordings of KCNN3 channel-expressing CHO cells demonstrated that disease-associated mutations result in gain of function of the mutant channels, characterized by increased Ca2+ sensitivity leading to faster and more complete activation of KCNN3 mutant channels. Pretreatment of cells with the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole revealed basal inhibition of wild-type and mutant KCNN3 channels by CK2. Analogous experiments with the KCNN3 p.Val450Leu mutant previously identified in a family with portal hypertension indicated basal constitutive channel activity and thus a different gain-of-function mechanism compared to the ZLS-associated mutant channels. With the report on de novo KCNK4 mutations in subjects with facial dysmorphism, hypertrichosis, epilepsy, ID, and gingival overgrowth, we propose to combine the phenotypes caused by mutations in KCNH1, KCNK4, and KCNN3 in a group of neurological potassium channelopathies caused by an increase in K+ conductance.

Ether-à-go-go K+ channels: effective modulators of neuronal excitability.

Mammalian ether-à-go-go (EAG) channels are voltage-gated K+ channels. They are encoded by the KCNH gene family and divided into three subfamilies, eag (Kv10), erg (eag-related gene; Kv11) and elk (eag-like; Kv12). All EAG channel subtypes are expressed in the brain where they effectively modulate neuronal excitability. This Topical Review describes the biophysical properties of each of the EAG channel subtypes, their function in neurons and the neurological diseases induced by EAG channel mutations. In contrast to the function of erg currents in the heart, where they contribute to repolarization of the cardiac action potential, erg currents in neurons are involved in the maintenance of the resting potential, setting of action potential threshold and frequency accommodation. They can even support high frequency firing by preventing a depolarization-induced Na+ channel block. EAG channels are modulated differentially, e.g. eag channels by intracellular Ca2+ , erg channels by extracellular K+ and GPCRs, and elk channels by changes in pH. So far, only currents mediated by erg channels have been recorded in neurons with the help of selective blockers. Neuronal eag and elk currents have not been isolated due to the lack of suitable channel blockers. However, findings in KO mice indicate a physiological role of eag1 currents in synaptic transmission and an involvement of elk2 currents in cognitive performance. Human eag1 and eag2 gain-of-function mutations underlie syndromes associated with epileptic seizures.

Marine compound rhizochalinin shows high in vitro and in vivo efficacy in castration resistant prostate cancer.

Development of drug resistance is an inevitable phenomenon in castration-resistant prostate cancer (CRPC) cells requiring novel therapeutic approaches. In this study, efficacy and toxicity of Rhizochalinin (Rhiz) - a novel sphingolipid-like marine compound - was evaluated in prostate cancer models, resistant to currently approved standard therapies. In vitro activity and mechanism of action of Rhiz were examined in the human prostate cancer cell lines PC-3, DU145, LNCaP, 22Rv1, and VCaP. Rhiz significantly reduced cell viability at low micromolar concentrations showing most pronounced effects in enzalutamide and abiraterone resistant AR-V7 positive cells. Caspase-dependent apoptosis, inhibition of pro-survival autophagy, downregulation of AR-V7, PSA and IGF-1 expression as well as inhibition of voltage-gated potassium channels were identified as mechanisms of action. Remarkably, Rhiz re-sensitized AR-V7 positive cells to enzalutamide and increased efficacy of taxanes.In vivo activity and toxicity were evaluated in PC-3 and 22Rv1 NOD SCID mouse xenograft models using i.p. administration. Rhiz significantly reduced growth of PC-3 and 22Rv1 tumor xenografts by 27.0% (p = 0.0156) and 46.8% (p = 0.047) compared with controls with an increased fraction of tumor cells showing apoptosis secondary to Rhiz exposure. In line with the in vitro data, Rhiz was most active in AR-V7 positive xenografts in vivo. In animals, no severe side effects were observed.In conclusion, Rhiz is a promising novel marine-derived compound characterized by a unique combination of anticancer properties. Its further clinical development is of high impact for patients suffering from drug resistant prostate cancer especially those harboring AR-V7 mediated resistance to enzalutamide and abiraterone.

Effects of Temperature on Heteromeric Kv11.1a/1b and Kv11.3 Channels.

Kv11.1 channels are crucial in cardiac physiology, and there is increasing evidence of physiological roles of different Kv11 channels outside the heart. The HERG (human Kv11.1a) channel has previously been shown to carry substantially more current at elevated temperatures, and we have now comparably investigated the temperature dependence of neuronal Kv11.3 channels and the more ubiquitous heteromeric Kv11.1a/1b channels. Transiently expressed rat Kv11 channels were studied at 21°C, 30°C, and 35°C. At near-physiological temperature, the maximal sustained outward current density was almost three times the mean value obtained at room temperature for Kv11.1a/1b, and increased by ∼150% for Kv11.3. For both channels, reduced inactivation contributed to the current increase at higher temperature. Elevated temperature moved Kv11.1a/1b isochronal activation curves to more negative potentials, but shifted the potential of half-maximal Kv11.3 channel activation to more depolarized values and reduced its voltage sensitivity. Thus, increased temperature stabilized the open state over the closed state of Kv11.1a/1b channels and exerted the opposite effect on Kv11.3 channel activation. Both Kv11 channels exhibited an overall high temperature sensitivity of most gating parameters, with remarkably high Q10 factors of ∼5 for the rate of Kv11.1a/1b activation. The Q10 factors for Kv11.3 gating were more uniform, but still higher for activation than for inactivation kinetics. The results demonstrate that characteristic differences between Kv11.1a/1b and Kv11.3 determined at room temperature do not necessarily apply to physiological conditions. The data provided here can aid in the design of models that will enhance our understanding of the role of Kv11 currents in excitable cells.

Mutations in KCNH1 and ATP6V1B2 cause Zimmermann-Laband syndrome.

Zimmermann-Laband syndrome (ZLS) is a developmental disorder characterized by facial dysmorphism with gingival enlargement, intellectual disability, hypoplasia or aplasia of nails and terminal phalanges, and hypertrichosis. We report that heterozygous missense mutations in KCNH1 account for a considerable proportion of ZLS. KCNH1 encodes the voltage-gated K(+) channel Eag1 (Kv10.1). Patch-clamp recordings showed strong negative shifts in voltage-dependent activation for all but one KCNH1 channel mutant (Gly469Arg). Coexpression of Gly469Arg with wild-type KCNH1 resulted in heterotetrameric channels with reduced conductance at positive potentials but pronounced conductance at negative potentials. These data support a gain-of-function effect for all ZLS-associated KCNH1 mutants. We also identified a recurrent de novo missense change in ATP6V1B2, encoding the B2 subunit of the multimeric vacuolar H(+) ATPase, in two individuals with ZLS. Structural analysis predicts a perturbing effect of the mutation on complex assembly. Our findings demonstrate that KCNH1 mutations cause ZLS and document genetic heterogeneity for this disorder.

"Alcohol and nicotine"--Concept and evaluation of an interdisciplinary elective course with OSPE in preclinical medical education.

In the last decade, increasing interest has been paid to interdisciplinary and practical courses in the medical education in Germany. This report describes the implementation and outcome of a preclinical interdisciplinary elective course with a team-teaching concept developed by lecturers in medical psychology, anatomy, physiology and biochemistry. The practical orientation of the course led to the implementation of a final interdisciplinary OSPE to ensure fair consideration of the different disciplines involved in grading. Individual OSPE results correlate well with the fact that different skills are required in medical psychology compared to those required in anatomy, physiology and biochemistry. Student course evaluation and lecturers` experience indicate the success of this elective course. Its concept can be well adapted to other interdisciplinary courses.

Effects of the small molecule HERG activator NS1643 on Kv11.3 channels.

NS1643 is one of the small molecule HERG (Kv11.1) channel activators and has also been found to increase erg2 (Kv11.2) currents. We now investigated whether NS1643 is also able to act as an activator of Kv11.3 (erg3) channels expressed in CHO cells. Activation of rat Kv11.3 current occurred in a dose-dependent manner and maximal current increasing effects were obtained with 10 µM NS1643. At this concentration, steady-state outward current increased by about 80% and the current increase was associated with a significant shift in the voltage dependence of activation to more negative potentials by about 15 mV. In addition, activation kinetics were accelerated, whereas deactivation was slowed. There was no significant effect on the kinetics of inactivation and recovery from inactivation. The strong current-activating agonistic effect of NS1643 did not result from a shift in the voltage dependence of Kv11.3 channel inactivation and was independent from external Na(+) or Ca(2+). At the higher concentration of 20 µM, NS1643 induced clearly less current increase. The left shift in the voltage dependence of activation reversed and the voltage sensitivity of activation dramatically decreased along with a slowing of Kv11.3 channel activation. These data show that, in comparison to other Kv11 family members, NS1643 exerts distinct effects on Kv11.3 channels with especially pronounced partial antagonistic effects at higher concentration.

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.

Strong activation of ether-à-go-go-related gene 1 K+ channel isoforms by NS1643 in human embryonic kidney 293 and Chinese hamster ovary cells.

Two different mechanisms leading to increased current have been described for the small-molecule human ether-à-go-go-related gene (herg) activator NS1643 [1,3-bis-(2-hydroxy-5-trifluoromethylphenyl)-urea]. On herg1a channels expressed in Xenopus laevis oocytes, it mainly acts via attenuation of inactivation and for rat (r) erg1b channels expressed in human embryonic kidney (HEK)-293 cells, it strongly shifts the activation curve to the left. We now investigated the NS1643 effects on erg1b channels in more detail and performed comparative experiments with rat and human erg1a in different expression systems. Significant differences were observed between expression systems, but not between the rat and human isoform. In HEK-293 or Chinese hamster ovary (CHO) cells, activation of rat erg1b channels occurred in a dose-dependent manner with a maximum current increase of 300% obtained with 10 µM NS1643. In contrast, the NS1643-induced strong leftward shift in the voltage dependence of activation further increased with higher drug concentration, needed more time to develop, and exhibited use dependence. Coexpression of KCNE1 or KCNE2 did not attenuate this NS1643 effect on erg1 channel activation and did thus not mimic the lower drug potency on this parameter observed in oocytes. NS1643 (10 µM) slowed erg1b channel deactivation and recovery from inactivation without significant changes in activation and inactivation kinetics. With the exception of accelerated activation, NS1643 affected erg1a channels similarly, but the effect was less pronounced than in erg1b or erg1a/1b channels. It is noteworthy that rerg1b and herg1a inactivation estimated from fully activated current voltage relationships were unaltered in the continued presence of 10 µM NS1643 in the mammalian expression systems, indicating qualitative differences from NS1643 effects in X. laevis oocytes.

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.

Gonadotropin-releasing hormone inhibits ether-à-go-go-related gene K+ currents in mouse gonadotropes.

Secretion of LH from gonadotropes is initiated by a GnRH-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). This increase in [Ca(2+)](i) is the result of Ca(2+) release from intracellular stores and Ca(2+) influx through voltage-dependent Ca(2+) channels. Here we describe an ether-à-go-go-related gene (erg) K(+) current in primary mouse gonadotropes and its possible function in the control of Ca(2+) influx. To detect gonadotropes, we used a knock-in mouse strain, in which GnRH receptor-expressing cells are fluorescently labeled. Erg K(+) currents were recorded in 80-90% of gonadotropes. Blockage of erg currents by E-4031 depolarized the resting potential by 5-8 mV and led to an increase in [Ca(2+)](i), which was abolished by nifedipine. GnRH inhibited erg currents by a reduction of the maximal erg current and in some cells additionally by a shift of the activation curve to more positive potentials. In conclusion, the erg current contributes to the maintenance of the resting potential in gonadotropes, thereby securing a low [Ca(2+)](i) by restricting Ca(2+) influx. In addition, the erg channels are modulated by GnRH by an as-yet unknown signal cascade.

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.

Functional characterization of genetically labeled gonadotropes.

Gonadotropes are crucial in the control of reproduction but difficult to isolate for functional analysis due to their scattered distribution in the anterior pituitary gland. We devised a binary genetic approach, and describe a new mouse model that allows visualization and manipulation of gonadotrope cells. Using gene targeting in embryonic stem cells, we generated mice in which Cre recombinase is coexpressed with the GnRH receptor, which is expressed in gonadotrope cells. We show that we can direct Cre-mediated recombination of a yellow fluorescent protein reporter allele specifically in gonadotropes within the anterior pituitary of these knock-in mice. More than 99% of gonadotropin-containing cells were labeled by yellow fluorescent protein fluorescence and readily identifiable in dissociated pituitary cell culture, allowing potentially unbiased sampling from the gonadotrope population. Using electrophysiology, calcium imaging, and the study of secretion on the single-cell level, the functional properties of gonadotropes isolated from male mice were analyzed. Our studies demonstrate a significant heterogeneity in the resting properties of gonadotropes and their responses to GnRH. About 50% of gonadotropes do not exhibit secretion of LH or FSH. Application of GnRH induced a broad range of both electrophysiological responses and increases in the intracellular calcium concentration. Our mouse model will also be able to direct expression of other Cre recombination-dependent reporter genes to gonadotropes and, therefore, represents a versatile new tool in the understanding of gonadotrope biology.

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.

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.

Extracellular potassium effects are conserved within the rat erg K+ channel family.

The biophysical properties of native cardiac erg1 and recombinant HERG1 channels have been shown to be influenced by the extracellular K(+) concentration ([K(+)](o)). The erg1 conductance, for example, increases dramatically with a rise in [K(+)](o). In the brain, where local [K(+)](o) can change considerably with the extent of physiological and pathophysiological neuronal activity, all three erg channel subunits are expressed. We have now investigated and compared the effects of an increase in [K(+)](o) from 2 to 10 mm on the three rat erg channels heterologously expressed in CHO cells. Upon increasing [K(+)](o), the voltage dependence of activation was shifted to more negative potentials for erg1 (DeltaV(0.5) = -4.0 +/- 1.1 mV, n = 28) and erg3 (DeltaV(0.5) = -8.4 +/- 1.2 mV, n = 25), and was almost unchanged for erg2 (DeltaV(0.5) = -2.0 +/- 1.3 mV, n = 6). For all three erg channels, activation kinetics were independent of [K(+)](o), but the slowing of inactivation by increased [K(+)](o) was even more pronounced for erg2 and erg3 than for erg1. In addition, with increased [K(+)](o), all three erg channels exhibited significantly slower time courses of recovery from inactivation and of deactivation. Whole-cell erg-mediated conductance was determined at the end of 4 s depolarizing pulses as well as with 1 s voltage ramps starting from the fully activated state. The rise in [K(+)](o) resulted in increased conductance values for all three erg channels which were more pronounced for erg2 (factor 3-4) than for erg1 (factor 2.5-3) and erg3 (factor 2-2.5). The data demonstrate that most [K(+)](o)-dependent changes in the biophysical properties are well conserved within the erg K(+) channel family, despite gradual differences in the magnitude of the effects.

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.

Functions of erg K+ channels in excitable cells.

Ether-à-go-go-related gene (erg) channels are voltage-dependent K+ channels mediating inward-rectifying K+ currents because of their peculiar gating kinetics. These characteristics are essential for repolarization of the cardiac action potential. Inherited and acquired malfunctioning of erg channels may lead to the long QT-syndrome. However, erg currents have also been recorded in many other excitable cells, like smooth muscle fibres of the gastrointestinal tract, neuroblastoma cells or neuroendocrine cells. In these cells erg currents contribute to the maintenance of the resting potential. Changes in the resting potential are related to cell-specific functions like increase in hormone secretion, frequency adaptation or increase in contractility.