The Antibody Targeting the E314 Peptide of Human Kv1.3 Pore Region Serves as a Novel, Potent and Specific Channel Blocker

Selective blockade of Kv1.3 channels in effector memory T (TEM) cells was validated to ameliorate autoimmune or autoimmune-associated diseases. We generated the antibody directed against one peptide of human Kv1.3 (hKv1.3) extracellular loop as a novel and possible Kv1.3 blocker. One peptide of hKv1.3 extracellular loop E3 containing 14 amino acids (E314) was chosen as an antigenic determinant to generate the E314 antibody. The E314 antibody specifically recognized 63.8KD protein stably expressed in hKv1.3-HEK 293 cell lines, whereas it did not recognize or cross-react to human Kv1.1(hKv1.1), Kv1.2(hKv1.2), Kv1.4(hKv1.4), Kv1.5(hKv1.5), KCa3.1(hKCa3.1), HERG, hKCNQ1/hKCNE1, Nav1.5 and Cav1.2 proteins stably expressed in HEK 293 cell lines or in human atrial or ventricular myocytes by Western blotting analysis and immunostaining detection. By the technique of whole-cell patch clamp, the E314 antibody was shown to have a directly inhibitory effect on hKv1.3 currents expressed in HEK 293 or Jurkat T cells and the inhibition showed a concentration-dependence. However, it exerted no significant difference on hKv1.1, hKv1.2, hKv1.4, hKv1.5, hKCa3.1, HERG, hKCNQ1/hKCNE1, L-type Ca2+ or voltage-gated Na+ currents. The present study demonstrates that the antibody targeting the E314 peptide of hKv1.3 pore region could be a novel, potent and specific hKv1.3 blocker without affecting a variety of closely related Kv1 channels, KCa3.1 channels and functional cardiac ion channels underlying central nervous systerm (CNS) disorders or drug-acquired arrhythmias, which is required as a safe clinic-promising channel blocker

Potassium Channel Block by a Tripartite Complex of Two Cationophilic Ligands and a Potassium IonS⃞

Voltage-gated potassium channels (Kv) are targets for drugs of large chemical diversity. Although hydrophobic cations block Kv channels with Hill coefficients of 1, uncharged electron-rich (cationophilic) molecules often display Hill coefficients of 2. The mechanism of the latter block is unknown. Using a combination of computational and experimental approaches, we mapped the receptor for the immunosuppressant PAP-1 (5-(4-phenoxybutoxy)psoralen), a high-affinity blocker of Kv1.3 channels in lymphocytes. Ligand-docking using Monte Carlo minimizations suggested a model in which two cationophilic PAP-1 molecules coordinate a K+ ion in the pore with their coumarin moieties, whereas the hydrophobic phenoxyalkoxy side chains extend into the intrasubunit interfaces between helices S5 and S6. We tested the model by generating 58 point mutants involving residues in and around the predicted receptor and then determined their biophysical properties and sensitivity to PAP-1 by whole-cell patch-clamp. The model correctly predicted the key PAP-1-sensing residues in the outer helix, the P-loop, and the inner helix and explained the Hill coefficient of 2 by demonstrating that the Kv1.3 pore can accommodate two or even four PAP-1 molecules. The model further explained the voltage-dependence of block by PAP-1 and its thousand-fold selectivity for Kv1.3 over non-Kv1 channels. The 23- to 125-fold selectivity of PAP-1 for Kv1.3 over other Kv1 channels is probably due to its preferential affinity to the C-type inactivated state, in which cessation of K+ flux stabilizes the tripartite PAP-1:K+:PAP-1 complex in the pore. Our study provides a new concept for potassium channel block by cationophilic ligands