Background: Certain types of potassium channels (known as Eag1, KCNH1, Kv10.1) are associated with the production of tumours in patients and in animals. We have now studied the expression pattern of the Eag1 channel in a large range of normal and tumour tissues from different collections utilising molecular biological and immunohistochemical techniques.

The potassium channel ether à go-go has been directly linked to cellular proliferation and transformation, although its physiologic role(s) are as of yet unknown. The specific blockade of human Eag1 (hEag1) may not only allow the dissection of the role of the channel in distinct physiologic processes, but because of the implication of hEag1 in tumor biology, it may also offer an opportunity for the treatment of cancer. However, members of the potassium channel superfamily are structurally very similar to one another, and it has been notoriously difficult to obtain specific blockers for any given channel. Here, we describe and validate the first rational design of a monoclonal antibody that selectively inhibits a potassium current in intact cells. Specifically blocking hEag1 function using this antibody inhibits tumor cell growth both in vitro and in vivo. Our data provide a proof of concept that enables the generation of functional antagonistic monoclonal antibodies against ion channels with therapeutic potential. The particular antibody described here, as well as the technique developed to make additional functional antibodies to Eag1, makes it possible to evaluate the potential of the channel as a target for cancer therapy. [Cancer Res 2007;67(15):7343-9]

R.M.Weseloh and L.A.Pardo contributed equally to this workA short C-terminal domain is required for correct tetrameric assembly in some potassium channels. Here, we show that this domain forms a coiled coil that determines not only the stability but also the selectivity of the multimerization. Synthetic peptides comprising the sequence of this domain in Eag1 and other channels are able to form highly stable tetrameric coiled coils and display selective heteromultimeric interactions. We show that loss of function caused by disruption of this domain in Herg1 can be rescued by introducing the equivalent domain from Eag1, and that this chimeric protein can form heteromultimers with Eag1 while wild-type Erg1 cannot. Additionally, a short endoplasmic reticulum retention sequence closely preceding the coiled coil plays a crucial role for surface expression. Both domains appear to co-operate to form fully functional channels on the cell surface and are a frequent ®nding in ion channels. Many pathological phenotypes may be attributed to mutations affecting one or both domains.

No abstract

We have studied the properties of r-eag voltage-activated potassium channels in a stably transfected human embryonic kidney cell line. It was found that r-eag channels are rapidly and reversibly inhibited by a rise in intracellular calcium from 30 to 300 nM. The inhibition does not appear to depend on the activity of calcium-dependent kinases and phosphatases. The effect of calcium on r-eag channel activity was studied in inside-out membrane patches. Calcium inhibited r-eag channel activity with a mean IC50 Of 67 nM. Activation of muscarinic receptors, generating calcium oscillations in the transfected cells, induced a synchronous inhibition of r-eag mediated outward currents. This shows that calcium can mediate r-eag current inhibition following muscarinic receptor activation. The data indicate that r-eag channels are calcium-inhibitable voltage-activated potassium channels.The excitability of neurons is closely linked to the activity of potassium (K) channels. These ion channels are members of evolutionarily conserved multigene families (1-3). Recently, a distinct family of K channel genes related to the Drosophila ether-a'-go-go (eag) gene has emerged (4, 5). Sequence alignments of eag relatives from Drosophila, mouse, rat, and human have indicated that these K channels are structurally related both to voltage-activated K channels in the Shaker family and to cyclic nucleotide-gated cation channels. Heterologous expression of cloned members of the eag family demonstrated that the eag relatives express voltage-activated K channels with remarkably diverse electrophysiological properties (6)(7)(8)22).The Drosophila eag polypeptide mediated, in the Xenopus oocyte expression system, potassium-outward and calciuminward currents dependent on voltage and cAMP (6). The homologous rat eag (r-eag) polypeptide gave rise to voltageactivated K channels with rather different properties in comparison to Drosophila eag channels and other voltage-activated K channels (7,8). Most notably, activation kinetics of r-eag channels were dramatically altered by the holding membrane potential. Also, unlike Drosophila eag channels, r-eag channels did not mediate calcium-inward currents and were not modulated by cAMP. We now report that the induction of Ca2+ transients by the activation of muscarinic receptors was accompanied by a total transient block of r-eag currents in 293 cells stably transfected with r-eag cDNA. The Ca2+ inhibition was found to be rapid and reversible and was not mediated by calcineurin or Ca2+-dependent kinases. In inside-out patches, we observed an exquisite Ca2+ sensitivity, channel activity being almost completely suppressed by cytosolic Ca2+ concentrations above 150 nM. These data indicate that r-eag channels are Ca2+-inhibitable voltage-activated K channels. Thus, in neurons, they might specifically amplify excitatory stimuli related to membrane depolarization and elevation of intracellular Ca2+.