Overexpression of Eag1 potassium channels in clinical tumours.

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. RESULTS: The use of reverse transcription real-time PCR and specifically generated monoclonal anti-Eag1 antibodies showed that expression of the channel is normally limited to specific areas of the brain and to restricted cell populations throughout the body. Tumour samples, however, showed a significant overexpression of the channel with high frequency (up to 80% depending on the tissue source) regardless of the detection method (staining with either one of the antibodies, or detection of Eag1 RNA). CONCLUSION: Inhibition of Eag1 expression in tumour cell lines reduced cell proliferation. Eag1 may therefore represent a promising target for the tailored treatment of human tumours. Furthermore, as normal cells expressing Eag1 are either protected by the blood-brain barrier or represent the terminal stage of normal differentiation, Eag1 based therapies could produce only minor side effects.

Secondary structure of antisauvagine analogues is important for CRF receptor antagonism: development of antagonists with increased potency and receptor selectivity.

Antisauvagine-30 (aSVG) is the only high-affinity antagonist for the corticotropin-releasing factor (CRF) type 2 (CRF(2)) receptor. A structure-activity relationship study was performed to pinpoint residues conferring aSVG's selectivity. The aSVG-analogues being N-terminally extended by one or two residues or containing the Ala(22)Arg(23)Ala(24) (ARA-motif) of CRF, were synthesized. Additionally, a lactam bridge between positions 29 and 32 was introduced. The modified peptides were analyzed for alpha-helicity properties, binding affinities and antagonistic potencies at the rat CRF(1) and mouse CRF(2B) receptors. While N-terminal prolongation and replacement of D-Phe(11) by Tyr(11) increased the affinity for the CRF(2) receptor, the introduction of the ARA motif resulted in a loss of CRF(2) receptor selectivity. These data show that aSVG(10-40) analogues are more potent CRF(2) receptor antagonists than aSVG(11-40) peptides, while introduction of the ARA-motif or a cyclic constraint between residues 29 and 32 favors binding to the CRF(1) receptor.

C-terminal domains implicated in the functional surface expression of potassium channels.

A 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 finding in ion channels. Many pathological phenotypes may be attributed to mutations affecting one or both domains.