CaV1.3 C-terminal protein-protein interaction drugability

Voltage-gated Ca2+ channels mediate the entry of Ca2+ ions into electrically excitable cells during membrane depolarization. The resulting Ca2+ signal triggers many important physiological processes. During the last years we found that the so-called CaV 1.3 L-type calcium channels (LTCCs) contribute to a variety of physiological functions (including mood behavior, hearing, heart rate control and neuronal pacemaking in dopaminergic neurons) making them suitable drug targets. The aim of this project is to assess the potential of a novel regulatory mechanism that tightly controls the function of L-type Ca2+ channels (LTCCs) for the specific modulation of these channels. An intramolecular protein interaction between the distal portion of the C-terminal tail of CaV 1.3 LTCCs and more proximal C-terminal regions strongly modulates the Ca2+- and voltage-dependent gating of these channels. In the proposed project we want to determine whether interference with this intramolecular interaction could provide a novel pharmacological approach to modulate the CaV 1.3 channel activity also for therapeutic intervention. As a proof-of-concept, we attempt to develop short CaV 1.3-specific inhibitory peptides for this C- terminal interaction.

In FWF project P22528 we aimed to determine whether pharmacological interference with a C-terminal intramolecular interaction could provide a novel approach to modulate the Cav1.3 L-type calcium channel (LTCC) activity. We have sucessfully investigated a novel Cav1.3 isoform that is characterized by the absence of a C-terminal modulatory domain (CTM) that resides in the very distal C-terminus in the long form. To test the interference with this CTM as a novel strategy to modulate Cav1.3 LTCC activity by interference with the C-terminal gating modulator we have successfully setup a PCA-assay based on the reporter enzyme Renilla reniformis and an additional NMR-based protein-protein interaction reporter system. The project changed its focus in the second year of the funding period because I was appointed Professor of Molecular Sensory Physiology and Pharmacology at the Medical University of Vienna in March 2011. We extended our work on Cav1.4 LTCCs which are predominantly expressed in the retina. Due to our existing knowledge of the CTM we were able to functionally rescue the mutant phenotype by co-expression of the Cav1.4 CTM in a heterologous expression system. Further our analysis of two representative Cav1.4 mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches. Proper function of Cav1.4 L-type calcium channels is crucial for neurotransmitter release in the retina. Our understanding about how different levels of Cav1.4 channel activity affect retinal function is still limited. We could show that both loss and gain in Cav1.4 channel activity resulted in similar morphological alterations of the retina. Whether such similarity also applies on the functional level was yet to be elicited. Obviously the environment that Cav1.4 LTCC proteins face physiologically in the retina is much more complex. We have therefore set up a functional test based on a multielectrode array system that allows simultaneous recordings of retinal activity upon light stimulation (the physiological stimulus in the retinal system) to test the effect of different Cav1.4 mutations in its physiological environment. We already started with first analyses of isolated retinas from appropriate mouse models but this work is still in progress and will be continued in the course of FWF Project P-26881.