Cav1.3 L-type calcium channel dysfunction in human disease

Voltage-gated calcium-channels open upon plasma membrane depolarization of electrically excitable cells. They are calcium ion selective pores and therefore crucial determinants of calcium ion entry into cells. Their calcium-signal controls different physiological processes, including muscle contraction, hearing, heart beat and CNS function. L-type calcium-channels (LTCCs) 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. Four different L-type calcium channels isoforms exist which mediate different physiological functions. Cav1.2 and Cav1.3 are the most abundantly expressed isoforms in most cells. Aim of this project is to assess how changes in the activity of Cav1.3 LTCCs affect physiological functions and how this relates to human disease. This work builds on our recent findings of novel modulatory domains within the pore forming subunit of these channels and of our recent discovery of human Cav1.3 gain of function mutations. During several FWFfunded research projects over the past 17 years our laboratory has established the role of Cav1.3 LTTCs for physiology in rodents with most insight gained by the selective genetic or pharmacological inhibition of these. We also established their predominant role for sinoatrial-node pacemaking and hearing in humans. A novel challenge is now to understand how subtle changes, including hyperactivity, of these channels affect specific cellular functions and contribute to disease. This aspect is of therapeutic interest because LTCC blockers in clinical use as antihypertensives could be further developed for other diseases with inappropriate Cav1.3 activity. Towards this aim we can now take advantage of a novel unique mouse model which we have successfully generated and validated in a recent project. These mice express Cav1.3 channels in which regulation by C- terminal alternative splicing is prevented. This allows us to determine the consequences of abnormal, splicing-dependent subtle functional changes of Cav1.3 in vivo and in vitro. We can also build on our recently discovered human gain of function mutations associated with hyperaldosteronism and a complex multi-organ syndrome. One of these human mutations will serve to generate mutant mice in order to reveal the pathophysiological role of hyperactive Cav1.3 channels in the mammalian organism. This will also enable us to better understand the complex phenotype previously reported in patients with this germline mutation. Our work is expected to also provide us with novel insight into the role of Cav1.3 hyperactivity in neurodevelopmental disorders, including central auditory processing disorders and autism.

Voltage-gated calcium-channels open in response to stimulation in electrically excitable cells. They comprise calcium-selective ion pores permitting calcium ions to enter the cell, which controls a number of important physiological processes, including muscle contraction, cardiac pacemaking, hearing, endocrine and neuronal functions. L-type calcium channels are already an established target for so-called calcium channel blockers (such as amlodipine). These drugs are used worldwide for the treatment of high blood pressure, angina pectoris and cardiac arrhythmias. There are four different isoforms of L-type calcium-channels, Cav1.2 and Cav1.3 being the most common forms in most tissues. The aim of this research project was to find out how changes in the function of Cav1.3 channels influence physiological processes and how this leads to human diseases. In doing so, we built on previous research results from our group, in which we discovered that genetic diseases that result in the loss of the Cav1.3 calcium-channel protein are associated with hearing impairment and irregular heartbeat. In this project we were able to show that genetic diseases that lead to increased Cav1.3 calcium-channel activity can cause severe neuronal developmental disorders, often with additional hormonal defects. We have therefore developed an animal model in which we inserted a human mutation, which is responsible for autistic disorders and reduced intelligence in an 8-year-old girl, into the corresponding gene (cacna1d) of mice. These mice show many of the same human behavioral disorders as have been seen in other patients with such mutations. This model now enables us to test whether the administration of the above-mentioned calcium-channel blockers also leads to a reduction in symptoms in mice. This would then also justify the symptomatic treatment of such patients with these drugs. We were also able to demonstrate that Cav1.3-channels are somewhat less sensitive to these calcium-channel blockers than the Cav1.2 channels in the blood vessels, which are responsible for lowering blood pressure. However, using standardized in vitro tests, we were able to demonstrate that some of the mutants (including those in our mouse model) have an increased sensitivity to calcium channel blockers, but some also have a lower sensitivity. This enables us to select patients with those mutations who are more likely to respond to the therapy for clinical treatment trials. Furthermore, in this project we succeeded in elucidating a new and unexpected function of Cav1.3-channels. In the pacemaker cells of the heart, they are also responsible for the formation of a sodium channel that is important for the pacemaker function. In cells of the hearing organ, we were able to show that Cav1.3 calcium-channels associate with other proteins in order to stabilize certain biophysical properties that are considered essential for normal hearing.