Validation of KCa3.1 Channel Nifedipine Interaction Site Predicted by Rosetta Modeling Method

Abstract

Calcium-activated potassium channels, including the intermediate-conductance KCa3.1 channel and the related small-conductance KCa2 channels, monitor and regulate intracellular calcium and are ideal pharmacological targets for immunosuppression, fibroproliferative disorders, hypertension and various neurological diseases. However, the development of drugs specifically for these medically relevant channels faces serious challenges because there is no available crystal structure that could be used for structure-assisted drug design. We used the Kv1.2-Kv2.1 channel structure (pdb id: 2R9R) as a template to generate a homology model of the KCa3.1 channel transmembrane region using the Rosetta modeling method. Docking of small molecules that are known to block KCa3.1 channel currents using Rosetta generated structural models of channel - drug complexes that can be validated using experimental approaches. Here we report the validation of the binding sites for the dihydropyridine nifedipine predicted by Rosetta's lowest energy stimulation of the KCa3.1 channel complex. We used a combination of site-directed mutagenesis and electrophysiological patch-clamp recording to test the identified amino acid residues located between the pore lining S5 and S6 segment. Blocking of the KCa3.1 channel by nifedipine was reduced by altering the side chains at positions L209, T212 and V278 to either alanine or valine, with T212 the most affected residue. Bulkier side-chain change to phenylalanine confirmed T212 as the main interacting site for nifedipine without compromising the channel's affinity for TRAM-34. Thus, by correctly identified the interaction of the dihydropyridine nifedipine with the KCa3.1 channel complex, Rosetta models can be used to understand the molecular mechanism of action of KCa channel blockers and gating modulators and assist with drug design efforts.