Inhibitory effect of the C-terminal modulator domain of voltage-dependent calcium Cav1.3 channels on the channel gating and calcium-dependent inactivation by single-channel analysis of the native Cav1.3 splice variants

• Applicant: Dr. Elza Kuzmenkina
• Subject Area: Anatomy and Physiology ; Biophysics
• Term: from 2014 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 256584869

Due to activation at low potentials, voltage-dependent calcium Cav1.3 channels regulate cellular excitability and pacemaking (e.g., in central neurons and sinoatrial node). On the other hand, calcium entry at low thresholds can lead to calcium overload and result in pathological conditions. For example, the gain of Cav1.3 channel function is reported to be involved in the pathogenesis of Parkinson's and Alzheimer's diseases. The potentially toxic calcium influx through Cav1.3 channels is limited by a negative-feedback process, the calcium-dependent inactivation (CDI). The extent of the channel activity and CDI has to be finetuned for particular cellular needs. The C-terminal modulator (CTM) domain in the channel distal C-terminus shifts channel activation to more depolarized voltages and diminishes apparent CDI. Various CTM splice isoforms are expressed simultaneously in the brain. Whether they have separable functions is not clarified yet. Using a single-channel resolution of the patch-clamp method, we aim at characterizing functional differences among natural Cav1.3 splice variants. The influence of CTM on the different conformational states of the channel can be studied in detail by Markov modeling of the single-channel data. Our second aim is to elucidate the mechanism of CDI inhibition by CTM. It was proposed that intramolecular binding of CTM to the proximal C-terminus competes with the binding of calmodulin, a protein that senses calcium concentration for CDI. However, our preliminary single-channel data suggest that the substantial part of the CDI reduction may arise secondarily: as a result of the inhibition of the channel activation so that the population of states from which CDI occurs is decreased. With CDI modeling, we want to uncouple CTM effects on the channel activation gating and CDI. Further single-channel experiments with calmodulin not binding calcium will provide us with additional information on the channel gating at the fast phase, before CDI occurs. Thus, in this project, we strive to characterize, which regulatory bandwidth and mechanisms are provided by the diversity of CTM splice isoforms in order to shape calcium entry for specific cellular requirements.

• DFG Programme: Research Grants
• International Connection: Austria, USA
• Participating Persons: Professor Dr. Stefan Herzig; Dr. Jan Matthes; Professor Dr. Jörg Striessnig; Professor David T. Yue, Ph.D.