Human α3ß4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells.

BACKGROUND: The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the α3 and ß4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the α5 and/or ß2 subunits. We compared the properties of human α3ß4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and α:ß subunit ratio. METHODOLOGY/PRINCIPAL FINDINGS: Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the α subunit, as reported for α4ß2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9' of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike α4ß2 channels, for α3ß4 receptors the putative two-α form is the predominant one in oocytes (at 1:1 α:ß cRNA ratio). This two-α form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-α form is the predominant one in HEK cells transfected with a 1:1 α:ß DNA ratio or in oocytes at 9:1 α:ß RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-α form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-α form gave rise to short bursts (14 ms) of high conductance (39 pS). CONCLUSIONS/SIGNIFICANCE: Like other neuronal nicotinic receptors, the α3ß4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected.

Expression pattern of the ether-a-go-go-related (ERG) family proteins in the adult mouse central nervous system: evidence for coassembly of different subunits.

Voltage-dependent K+ channels are the main determinants in controlling cellular excitability within the central nervous system. Among voltage-dependent K+ channels, the ERG subfamily is deeply involved in the control of cellular excitability, both in mammals and in invertebrates. ERG channels are encoded by different genes: the erg1 gene, which can generate two alternative transcripts (erg1a and erg1b), erg2 and erg3. The aim of the present study was to determine the expression pattern and cellular localization of ERG proteins (ERG1, ERG2, and ERG3) in the mouse CNS, differentiating, for the first time, the ERG1A and ERG1B isoforms. To this purpose, novel specific antibodies were raised against the various channel proteins and their specificity and immunoreactivity tested. It emerged that: 1) all the erg genes were indeed translated in neuronal tissue; 2) ERG proteins distribution in the mouse CNS often overlapped, and only in specific areas each ERG protein showed a distinct pattern of expression; and 3) ERG proteins were generally expressed in neuronal soma, but dendritic and/or white matter labeling could be detected in specific areas. The finding that ERG proteins often have an overlapping expression suggests that neuronal ERG currents could be determined, at least in part, by heterotetrameric ERG channels. This suggestion is demonstrated to occur for ERG1A/ERG1B by showing that the two isoforms coassemble in mouse brain.

Human ether-a-go-go-related gene 1 channels are physically linked to beta1 integrins and modulate adhesion-dependent signaling.

Adhesive receptors of the integrin family are primarily involved in cell-extracellular matrix adhesion. Additionally, integrins trigger multiple signaling pathways that are involved in cell migration, proliferation, survival, and differentiation. We previously demonstrated that the activation of integrins containing the beta(1) subunit leads to a selective increase in potassium currents carried by the human ether-a-go-go-related gene (hERG) channels in neuroblastoma and leukemia cells; this current activation modulates adhesion-dependent differentiation in these cells. We hypothesized that the cross-talk between integrins and hERG channels could be traced back to the assembly of a macromolecular signaling complex comprising the two proteins. We tested this hypothesis in both SH-SY5Y neuroblastoma cells and in human embryonic kidney 293 cells stably transfected with hERG1 and, therefore, expressing only the full-length hERG1 protein on the plasma membrane. The beta(1) integrin and hERG1 coprecipitate in these cells and colocalize in both intracellular and surface membrane compartments. The two proteins also coprecipitate with caveolin-1, suggesting the localization of the complex in lipid rafts/caveolae. hERG1-transfected cells undergo an activation of hERG currents after beta(1) integrin-mediated adhesion to fibronectin; concomitant with this activation, the focal adhesion kinase associates with the hERG1 protein and becomes tyrosine phosphorylated. Using hERG1-specific inhibitors, we show that the tyrosine phosphorylation of focal adhesion kinase is strictly dependent on hERG channel activity. Similarly, the activity of the small GTPase Rac1 turned out to be dependent on hERG currents. On the whole, these data indicate that the hERG1 protein associates with beta(1) integrins and modulates adhesion receptor signaling.

herg1 gene and HERG1 protein are overexpressed in colorectal cancers and regulate cell invasion of tumor cells.

The acquisition of the capacity to invade surrounding tissues confers a more malignant phenotype to tumor cells and is necessary for the establishment of metastases. The understanding of the molecular mechanisms underlying cell invasion in human solid tumors such as colorectal cancers could provide not only more sensitive prognostic analyses but also novel molecular targets for cancer therapy. We report in this article that K(+) ion channels belonging to the HERG family are important determinants for the acquisition of an invasive phenotype in colorectal cancers. The herg1 gene and HERG1 protein are expressed in many colon cancer cell lines, and the activity of HERG channels modulates colon cancer cell invasiveness. Moreover, the amount of HERG1 protein expressed on the plasma membrane is directly related to the invasive phenotype of colon cancer cells. Finally, both the herg1 gene and HERG1 protein were expressed in a high percentage of primary human colorectal cancers, with the highest incidence occurring in metastatic cancers, whereas no expression could be detected either in normal colonic mucosa or in adenomas.

Inhibitory actions of the selective serotonin re-uptake inhibitor citalopram on HERG and ventricular L-type calcium currents.

Using whole-cell patch clamp recording of heterologous HERG-mediated currents in transfected mammalian cells, we observed that the selective serotonin re-uptake inhibitor citalopram blocks HERG with an IC(50) of 3.97 microM. This is slightly less potent than fluoxetine in our system (IC(50) of 1.50 microM). In isolated guinea pig ventricular cardiomyocytes citalopram inhibited L-type calcium current (I(Ca,L)). The voltage dependence of I(Ca,L) inactivation in the presence of 100 microM citalopram was shifted significantly leftward. As a result, the I(Ca,L) 'window' in citalopram was found to be (a) smaller and (b) leftward-shifted compared to control. The effects of citalopram on both calcium current amplitude and the I(Ca,L) 'window' may help to explain citalopram's good cardiac safety profile, given its propensity to block HERG at excessive dosages.