Cell Cycle–related Changes in the Conducting Properties of r-eag K+ Channels

Release from arrest in G2 phase of the cell cycle causes profound changes in rat ether-à-go-go (r-eag) K+ channels heterologously expressed in Xenopus oocytes. The most evident consequence of the onset of maturation is the appearance of rectification in the r-eag current. The trigger for these changes is located downstream of the activation of mitosis-promoting factor (MPF). We demonstrate here that the rectification is due to a voltage-dependent block by intracellular Na+ ions. Manipulation of the intracellular Na+ concentration indicates that the site of Na+ block is located ∼45% into the electrical distance of the pore and is only present in oocytes undergoing maturation. Since the currents through excised patches from immature oocytes exhibited a fast rundown, we studied CHO-K1 cells permanently transfected with r-eag. These cells displayed currents with a variable degree of block by Na+ and variable permeability to Cs+. Partial synchronization of the cultures in G0/G1 or M phases of the cell cycle greatly reduced the variability. The combined data obtained from mammalian cells and oocytes strongly suggest that the permeability properties of r-eag K+ channels are modulated during cell cycle–related processes

Tumor cell-selective apoptosis induction through targeting of KV10.1 via bifunctional TRAIL antibody

<p>Abstract</p> <p>Background</p> <p>The search for strategies to target ion channels for therapeutic applications has become of increasing interest. Especially, the potassium channel K_V10.1 (Ether-á-go-go) is attractive as target since this surface protein is virtually not detected in normal tissue outside the central nervous system, but is expressed in approximately 70% of tumors from different origins.</p> <p>Methods</p> <p>We designed a single-chain antibody against an extracellular region of K_V10.1 (scFv62) and fused it to the human soluble TRAIL. The K_V10.1-specific scFv62 antibody -TRAIL fusion protein was expressed in CHO-K1 cells, purified by chromatography and tested for biological activity.</p> <p>Results</p> <p>Prostate cancer cells, either positive or negative for K_V10.1 were treated with the purified construct. After sensitization with cytotoxic drugs, scFv62-TRAIL induced apoptosis only in K_V10.1-positive cancer cells, but not in non-tumor cells, nor in tumor cells lacking K_V10.1 expression. In co-cultures with K_V10.1-positive cancer cells the fusion protein also induced apoptosis in bystander K_V10.1-negative cancer cells, while normal prostate epithelial cells were not affected when present as bystander.</p> <p>Conclusions</p> <p>K_V10.1 represents a novel therapeutic target for cancer. We could design a strategy that selectively kills tumor cells based on a K_V10.1-specific antibody.</p

The potassium channel Ether à go-go is a novel prognostic factor with functional relevance in acute myeloid leukemia

<p>Abstract</p> <p>Background</p> <p>The voltage-gated potassium channel hEag1 (K_V10.1) has been related to cancer biology. The physiological expression of the human channel is restricted to the brain but it is frequently and abundantly expressed in many solid tumors, thereby making it a promising target for a specific diagnosis and therapy. Because chronic lymphatic leukemia has been described not to express hEag1, it has been assumed that the channel is not expressed in hematopoietic neoplasms in general.</p> <p>Results</p> <p>Here we show that this assumption is not correct, because the channel is up-regulated in myelodysplastic syndromes, chronic myeloid leukemia and almost half of the tested acute myeloid leukemias in a subtype-dependent fashion. Most interestingly, channel expression strongly correlated with increasing age, higher relapse rates and a significantly shorter overall survival. Multivariate Cox regression analysis revealed hEag1 expression levels in AML as an independent predictive factor for reduced disease-free and overall survival; such an association had not been reported before. As a functional correlate, specific hEag1 blockade inhibited the proliferation and migration of several AML cell lines and primary cultured AML cells <it>in vitro</it>.</p> <p>Conclusion</p> <p>Our observations implicate hEag1 as novel target for diagnostic, prognostic and/or therapeutic approaches in AML.</p

Potassium channels as tumour markers

AbstractAn increasing number of ion channels are being found to be causally involved in diseases, giving rise to the new field of “channelopathies”. Cancer is no exception, and several ion channels have been linked to tumour progression. Among them is the potassium channel EAG (Ether-a-go-go). Over 75% of tumours have been tested positive using a monoclonal antibody specific for EAG, while inhibition of this channel decreased the proliferation of EAG expressing cells. The inhibition of EAG is accomplished using RNA interference, functional anti-EAG1 antibodies, or (unspecific) EAG channel blockers. Fluorescently labelled recombinant Fab fragments recognizing EAG allow the distribution of EAG to be visualized in an in vivo mouse tumour model

Ether à go-go potassium channel expression in soft tissue sarcoma patients

BACKGROUND: The expression of the human Eag1 potassium channel (Kv10.1) is normally restricted to the adult brain, but it has been detected in both tumour cell lines and primary tumours. Our purpose was to determine the frequency of expression of Eag1 in soft tissue sarcoma and its potential clinical implications. RESULTS: We used specific monoclonal antibodies to determine the expression levels of Eag1 in soft tissue sarcomas from 210 patients by immunohistochemistry. Eag1 was expressed in 71% of all tumours, with frequencies ranging from 56% (liposarcoma) to 82% (rhabdomyosarcoma). We detected differences in expression levels depending on the histological type, but no association was seen between expression of this protein and sex, age, grade or tumour size. Four cell lines derived from relevant sarcoma histological types (fibrosarcoma and rhabdomyosarcoma) were tested for Eag1 expression by real-time RT-PCR. We found all four lines to be positive for Eag1. In these cell lines, blockage of Eag1 by RNA interference led to a decrease in proliferation. CONCLUSION: Eag1 is aberrantly expressed in over 70% sarcomas. In sarcoma cell lines, inhibition of Eag1 expression and/or function leads to reduced proliferation. The high frequency of expression of Eag1 in primary tumours and the restriction of normal expression of the channel to the brain, suggests the application of this protein for diagnostic or therapeutic purposes

Mechanism of Block of hEag1 K+ Channels by Imipramine and Astemizole

Ether à go-go (Eag; KV10.1) voltage-gated K+ channels have been detected in cancer cell lines of diverse origin and shown to influence their rate of proliferation. The tricyclic antidepressant imipramine and the antihistamine astemizole inhibit the current through Eag1 channels and reduce the proliferation of cancer cells. Here we describe the mechanism by which both drugs block human Eag1 (hEag1) channels. Even if both drugs differ in their affinity for hEag1 channels (IC50s are ∼2 μM for imipramine and ∼200 nM for astemizole) and in their blocking kinetics, both drugs permeate the membrane and inhibit the hEag1 current by selectively binding to open channels. Furthermore, both drugs are weak bases and the IC50s depend on both internal an external pH, suggesting that both substances cross the membrane in their uncharged form and act from inside the cell in their charged forms. Accordingly, the block by imipramine is voltage dependent and antagonized by intracellular TEA, consistent with imipramine binding in its charged form to a site located close to the inner end of the selectivity filter. Using inside- and outside-out patch recordings, we found that a permanently charged, quaternary derivative of imipramine (N-methyl-imipramine) only blocks channels from the intracellular side of the membrane. In contrast, the block by astemizole is voltage independent. However, as astemizole competes with imipramine and intracellular TEA for binding to the channel, it is proposed to interact with an overlapping intracellular binding site. The significance of these findings, in the context of structure–function of channels of the eag family is discussed

Participação dos professores na escola

Na proximidade do século XXI, a Escola Portuguesa vive momentos de incerteza, de preocupação e desafio face ao futuro. Estamos perante uma sociedade que não se compadece com uma escola parada no tempo, mas, sim, exige uma escola activa, dinâmica e aberta ao meio. Pretende-se uma escola que desenvolva uma cultura de participação1, que saiba partilhar a educação com a família (principal entidade, responsável pela educação), com os trabalhadores não docentes, com a comunidade envolvente e assim todos possam contribuir para o desenvolvimento pleno e harmonioso da personalidade dos indivíduos, tornando-os cidadãos mais responsáveis e livres na sociedade. É este tipo de escola que é preconizada pela Lei de Bases (Lei nº46 / 86, de 14 de Outubro) e que exige uma mudança do Sistema Tradicional de Ensino. Por isso, a escola de hoje exige novas posturas, novas responsabilidades de todos os que nela intervêm e contribuem para uma melhoria do ensino, quer sejam professores, pais ou outros. Mas, o papel do professor, como principal impulsionador e dinamizador, é e será determinante para o sucesso de qualquer reforma do sistema educativo. O professor assume o papel primordial de dinamizador de participação e de mobilização de todos os outros intervenientes, no sentido de os levar a darem o seu contributo e a assumirem a sua cota parte de responsabilidade na educação, para que a escola possa realizar os seus objectivos. A escola é um local onde se trocam experiências, onde todos os que aí participam vivem um pouco ou grande parte da sua vida. Por isso, é imprescindível que cada um se sinta parte integrante dela. Assim, abordaremos a escola como um espaço de interacção, bem como as formas de participação dos seus intervenientes. Porque compreender a acção dos que fazem a escola leva-nos a conhecer os estatutos de cada membro e os papéis a eles associados, as normas organizacionais que orientam a interacção e o contributo de cada um para a prossecução das actividades. É na conjugação destes factores que se definem as formas de cada um estar na escola

Primary structure and functional expression of a high voltage activated calcium channel from rabbit lung

The complete amino acid sequence of the receptor for organic calcium channel blockers (CaCB) from rabbit lung has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CaCB-sensitive high voltage activated calcium channel in Xenopus oocytes

A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore

Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4-S5 linker). However, our recent work on channels disrupted in the S4-S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of KV10.1 revealed that the S4-S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use "split" channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in KV10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4-S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4-S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism