C-terminal modulation of Cav1.3 L-Type Ca2+ Channels

Voltage-gated calcium-channels are pores in the plasma membrane of excitable cells which open upon membrane depolarization and allow calcium ions to enter the cell. This calcium-signal controls different physiological processes, including muscle contraction and neuronal function. L-type calcium-channels in the cardiovascular system are a well established target for so-called calcium-channel blockers which are widely used clinically to treat cardiovascular diseases such as hypertension, angina and arrhythmias. However, different L-type channel subtypes (mainly Cav1.2 and Cav1.3) are also expressed in neurons where they play an important role for anxiety- and depression-like behavior (Cav1.3) as well as for fear (Cav1.3) and spatial (Cav1.2) memory. Moreover, the Cav1.3 isoform was found to play a key role for the development of Parkinson`s disease. Therefore they also appear to represent interesting drug targets. The aim of this project is to assess the physiological significance and pharmacotherapeutic potential of a novel modulatory mechanism that tightly controls the function of L-type Ca2+ channels (LTCCs). We originally discovered this modulation in the pore-forming 1-subunits of Cav1.4 LTCCs (Singh et al., Nature Neuroscience 9: 1108-1116, 2006) by showing that the distal portion of their C-terminal tail binds to upstream C-terminal regions and thereby strongly modulates the Ca2+- and voltage-dependent gating of these channels (C-terminal modulatory mechanism, CTM). We now discovered that a similar CTM exists in Cav1.3 LTCCs. It controls those Cav1.3 gating properties that are of immediate relevance for their unique physiological (sinoatrial node pacemaking, hearing, emotional behavior and fear memory) and pathophysiological role (susceptibility to neurodegeneration of nigrostriatal neurons in Parkinson`s Disease). This includes the typical negative activation range of Cav1.3 channels. In the proposed project we want to investigate the molecular basis and the physiological significance of this CTM, predict if channel fine-tuning is changed under pathological conditions with altered electrical excitability (such as epilepsy, ataxia, migraine and cardiac dysfunction) and determine whether pharmacological interference with CTM function could provide a novel approach to modulate channel activity for therapeutic intervention. We also propose the generation of a novel mouse model containing an inducible mutation to directly study the functional role of this CTM in vivo and predict its suitability as a potential new drug target.

Voltage-gated calcium-channels are pores in the plasma membrane of excitable cells which open upon membrane depolarization and allow calcium ions to enter the cell. This calcium-signal controls different physiological processes, including muscle contraction and neuronal function. L-type calcium-channels in the cardiovascular system are well established targets for so-called calcium-channel blockers which are widely used clinically to treat cardiovascular diseases such as hypertension, angina and arrhythmias. However, different L-type channel subtypes (mainly the so-called Cav1.2 and Cav1.3 isoforms) are also expressed in neurons where they play an important role for anxiety- and depression-like behavior (Cav1.3) as well as for fear (Cav1.3) and spatial (Cav1.2) memory. Moreover, the Cav1.3 isoform was found to play a key role for the development of Parkinson's disease. Therefore they also appear to represent interesting drug targets.The aim of this project was to assess the physiological significance of a novel modulatory mechanism that tightly controls the function of L-type Ca2+ channels (LTCCs). We originally discovered this modulation in the pore-forming ?1-subunits of Cav1.4 LTCCs (Singh et al., Nature Neuroscience 9: 1108-1116, 2006) by showing that the distal portion of their C-terminal tail binds to upstream C-terminal regions and thereby strongly modulates the Ca2+- and voltage-dependent gating of these channels (C-terminal modulatory mechanism, CTM). In this project we confirmed that a similar CTM exists in Cav1.3 LTCCs and that this is itself subject to modulation by alternative splicing. We showed that alternative splicing within the C-terminus generates channel variants that lack the CTM and therefore show different opening and closing behavior. Interestingly, we found that these splice variants are expressed in a highly tissue-dependent manner indicating that the properties of the channel are adjusted to specific cellular needs by alternative splicing (in addition to normal brain function the channel is known to be required for normal hearing and cardiac pacemaking). Quite unexpected was our discovery of a new human disease in which a gene mutation was found in an alternatively spliced exon in the channel pore of Cav1.3 LTCCs. The affected individuals are deaf and have a slow and arrhythmic heartbeat. We could show that the affected splice variant is the major one in heart and in the sensory cells of the inner ear and that the mutation prevents the (otherwise intact) channel from opening and allowing Ca2+ influx into the cell. We named this genetic disease SANDD (Sinoatrial Node Dysfunction and Deafness). As planned we also succeeded in constructing a mutant mouse in which we prevent the CTM from functioning. These mice are viable and we currently expand the colony of homozygous mutants for further studies in which we will determine the functional role of the CTM in vivo (especially on hearing, heart pacemaking, and brain function). The mice will be a fundamental biological tool to understand the physiological significance of Cav1.3 alternative splicing and the functioning of the CTM. If lack of the CTM turns out to protect animals from certain diseases (such as Parkinsons Disease) or shows any other therapeutically interesting phenotype (e.g. antidepressant-like effects), then efforts could be made to discover CTM-inhibitory molecules in upcoming projects.