Role of Neuronal Junctophilins in Recruitment and Modulation of Voltage-Gated Calcium Channels in PM-ER Junctions

Small conductance calcium activated potassium (SK) channels are predominantly expressed in heart atria and contribute to late repolarization of the action potential. Inhibition of SK current has been proposed as a therapy for atrial fibrillation (AF). We aim to develop an accurate model of SK channel gating and drug interaction that can be used to probe the role of SK channels in atrial health and disease. The model, based on Hirschberg et al's (1998) SK2 model, incorporates 4 closed and 2 open states. To generate a model with utility at physiologic temperature, we performed inside-out macropatch voltage clamp ramps at 23° and 37° C, and observed a pronounced leftward EC50 shift at 37° C (0.53 ± 0.07 µM and 0.38 ± 0.02 µM to 0.23 ± 0.02 µM and 0.28 ± 0.01 µM for hSK3 and hSK2, respectively). Thus, taking calcium activation of the major hSK isoforms into the diastolic calcium range. To provide a first-principles basis for constraining the kinetics of multi-step calcium-dependent activation, and binding of known and novel pharmacologic inhibitors, we performed single-channel recordings of SK2 at 23° C. Opening events without inhibitors were best fit with two major open-state conductances (low: 10.9 ± 1.6 pS, 60 ± 41% of events; high: 14.9 ± 1.4 pS, 40 ± 41% of events), justifying the multiple open states included in the model and to be further analysed regarding state specific kinetics. We will also present single channel data on known inhibitor apamin and novel modulator AP14145 constraining a pharmacological model. In conclusion, these experiments provided novel findings on temperature sensitivity of SK channels. These will be incorporated into an improved Markov model of SK channel dynamics to elucidate the roles of SK channels in health and disease.