No abstract

Peptide toxins from animal venom have been used for many years for the identification and study of cation-permeable ion channels. However, no peptide toxins have been identified that interact with known anion-selective channels, including cystic fibrosis transmembrane conductance regulator (CFTR), the protein defective in cystic fibrosis and a member of the ABC transporter superfamily. Here, we describe the identification and initial characterization of a novel 3.7-kDa peptide toxin, GaTx1, which is a potent and reversible inhibitor of CFTR, acting from the cytoplasmic side of the membrane. Thus, GaTx1 is the first peptide toxin identified that inhibits a chloride channel of known molecular identity. GaTx1 exhibited high specificity, showing no effect on a panel of nine transport proteins, including Cl ؊ and K ؉ channels, and ABC transporters. GaTx1-mediated inhibition of CFTR channel activity is strongly state-dependent; both potency and efficacy are reduced under conditions of elevated [ATP], suggesting that GaTx1 may function as a non-competitive inhibitor of ATP-dependent channel gating. This tool will allow the application of new quantitative approaches to study CFTR structure and function, particularly with respect to the conformational changes that underlie transitions between open and closed states.

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent K D of ϳ20 pM, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltagegated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.

Portugal is one of the most egalitarian countries in Europe in terms of lesbian, gay, bisexual, transgender, and intersex (LGBTI) individuals' legal rights. However, regarding education Portugal still lacks specific policies, plans and interventions to protect LGBTI students. To assess the perceptions of self-identified LGBTI youth regarding their school context, a total of 663 participants (aged from 15 to 20 years old) filled in an on-line questionnaire about their school climate. One hundred and forty-six of them answered an open-ended question about their personal experiences. A thematic analysis of these answers was conducted, and four main categories were identified: (i) victimization, (ii) coming out experiences, (iii) support networks, and (iv) demands. Most participants reported experiences of discrimination, and several sources of prejudice were identified. Furthermore, participants also recognized a lack of LGBTI information in school curriculum and made several demands. Besides inclusive laws, we suggest that the safety and the well-being of LGBTI youths in Portuguese schools depend upon others measures, such as teacher and school staff training, curricula inclusive of LGBTI diversity, and local strategies, such as Gay-Straight Alliances.