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

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

Dual role of protein kinase C on BK channel regulation

Large conductance voltage- and Ca2+-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 S695 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 S1151 in the C terminus of the channel α-subunit. Phosphorylation of only one α-subunit at S1151 and S695 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 S695. PKC phosphorylation at S1151 also influences stimulation of BK channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S1151A mutant channel is activated by PKA only, the phosphorylation of S1151 by PKC renders the channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S695 by PKC or introducing a phosphomimetic aspartate at this position (S695D) 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

Separation of M-like current and ERG current in NG108-15 cells

1. Differentiated NG108-15 neuroblastoma×glioma hybrid cells were whole-cell voltage-clamped. Hyperpolarizing pulses, superimposed on a depolarized holding potential (−30 or −20 mV), elicited deactivation currents which consisted of two components, distinguishable by fitting with two exponential functions. 2. Linopirdine [DuP 996, 3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one), a neurotransmitter-release enhancer known as potent and selective blocker of the M-current of rat sympathetic neurons, in concentrations of 5 or 10 μM selectively inhibited the fast component (IC(50)=14.7 μM). The slow component was less sensitive to linopirdine (IC(50)>20 μM). 3. The class III antiarrhythmics [(4-methylsulphonyl)amido]benzenesulphonamide (WAY-123.398) and 1-[2-(6-methyl-2-pyrydinil)ethyl]-4-(4-methylsulphonylaminobenzoyl) piperidine (E-4031), selective inhibitors of the inwardly rectifying ERG (ether-à-go-go-related gene) potassium channel, inhibited predominantly the slow component (IC(50)=38 nM for E-4031). The time constant of the WAY-123.398-sensitive current resembled the time constant of the slow component in size and voltage dependence. 4. Inwardly rectifying ERG currents, recorded in K(+)-rich bath at strongly negative pulse potentials, resembled the slow component of the deactivation current in their low sensitivity to linopirdine (28% inhibition at 50 μM). 5. The size of the slow component varied greatly between cells. Accordingly, varied the effect of WAY-123.398 on deactivation current and holding current. 6. RNA transcripts for the following members of the ether-à-go-go gene (EAG) K(+) channel family were found in differentiated NG108-15 cells: ERG1, ERG2, EAG1, EAG-like (ELK)1, ELK2; ERG3 was only present in non-differentiated cells. In addition, RNA transcripts for KCNQ2 and KCNQ3 were found in differentiated and non-differentiated cells. 7. We conclude that the fast component of the deactivation current is M-like current and the slow component is deactivating ERG current. The molecular correlates are probably KCNQ2/KCNQ3 and ERG1/ERG2, respectively