Alexandra Pinggera

L‐type Ca2+ channels in heart and brain

L-type calcium channels (Cav1) represent one of the three major classes (Cav1–3) of voltage-gated calcium channels. They were identified as the target of clinically used calcium channel blockers (CCBs; so-called calcium antagonists) and were the first class accessible to biochemical characterization. Four of the 10 known α1 subunits (Cav1.1–Cav1.4) form the pore of L-type calcium channels (LTCCs) and contain the high-affinity drug-binding sites for dihydropyridines and other chemical classes of organic CCBs. In essentially all electrically excitable cells one or more of these LTCC isoforms is expressed, and therefore it is not surprising that many body functions including muscle, brain, endocrine, and sensory function depend on proper LTCC activity. Gene knockouts and inherited human diseases have allowed detailed insight into the physiological and pathophysiological role of these channels. Genome-wide association studies and analysis of human genomes are currently providing even more hints that even small changes of channel expression or activity may be associated with disease, such as psychiatric disease or cardiac arrhythmias. Therefore, it is important to understand the structure–function relationship of LTCC isoforms, their differential contribution to physiological function, as well as their fine-tuning by modulatory cellular processes.

CACNA1D De Novo Mutations in Autism Spectrum Disorders Activate Cav1.3 L-Type Calcium Channels

Background Cav1.3 voltage-gated L-type calcium channels (LTCCs) are part of postsynaptic neuronal signaling networks. They play a key role in brain function, including fear memory and emotional and drug-taking behaviors. A whole-exome sequencing study identified a de novo mutation, p.A749G, in Cav1.3 α1-subunits (CACNA1D), the second main LTCC in the brain, as 1 of 62 high risk–conferring mutations in a cohort of patients with autism and intellectual disability. We screened all published genetic information available from whole-exome sequencing studies and identified a second de novo CACNA1D mutation, p.G407R. Both mutations are present only in the probands and not in their unaffected parents or siblings. Methods We functionally expressed both mutations in tsA-201 cells to study their functional consequences using whole-cell patch-clamp. Results The mutations p.A749G and p.G407R caused dramatic changes in channel gating by shifting (~15 mV) the voltage dependence for steady-state activation and inactivation to more negative voltages (p.A749G) or by pronounced slowing of current inactivation during depolarizing stimuli (p.G407R). In both cases, these changes are compatible with a gain-of-function phenotype. Conclusions Our data, together with the discovery that Cav1.3 gain-of-function causes primary aldosteronism with seizures, neurologic abnormalities, and intellectual disability, suggest that Cav1.3 gain-of-function mutations confer a major part of the risk for autism in the two probands and may even cause the disease. Our findings have immediate clinical relevance because blockers of LTCCs are available for therapeutic attempts in affected individuals. Patients should also be explored for other symptoms likely resulting from Cav1.3 hyperactivity, in particular, primary aldosteronism.

Splice variants of the CaV1.3 L-type calcium channel regulate dendritic spine morphology.

Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson’s disease. The L-type calcium channel CaV1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length CaV1.3L and two C-terminally truncated splice variants (CaV1.342A and CaV1.343S) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged CaV1.3L revealed its localization at the base-, neck-, and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in CaV1.3L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with CaV1.3L and correlated with increased CaV1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of CaV1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of CaV1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders.

Cav1.3 (CACNA1D) L-type Ca2+ channel dysfunction in CNS disorders

Cav1.3 belongs to the family of voltage‐gated L‐type Ca2+ channels and is encoded by the CACNA1D gene. Cav1.3 channels are not only essential for cardiac pacemaking, hearing and hormone secretion but are also expressed postsynaptically in neurons, where they shape neuronal firing and plasticity. Recent findings provide evidence that human mutations in the CACNA1D gene can confer risk for the development of neuropsychiatric disease and perhaps also epilepsy. Loss of Cav1.3 function, as shown in knock‐out mouse models and by human mutations, does not result in neuropsychiatric or neurological disease symptoms, whereas their acute selective pharmacological activation results in a depressive‐like behaviour in mice. Therefore it is likely that CACNA1D mutations enhancing activity may be disease relevant also in humans. Indeed, whole exome sequencing studies, originally prompted to identify mutations in primary aldosteronism, revealed de novo CACNA1D missense mutations permitting enhanced Ca2+ signalling through Cav1.3. Remarkably, apart from primary aldosteronism, heterozygous carriers of these mutations also showed seizures and neurological abnormalities. Different missense mutations with very similar gain‐of‐function properties were recently reported in patients with autism spectrum disorders (ASD). These data strongly suggest that CACNA1D mutations enhancing Cav1.3 activity confer a strong risk for – or even cause – CNS disorders, such as ASD.

A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.

Background: L-type Ca2+ channels (LTCCs) are fine-tuned by different molecular mechanisms. Results: Pore-forming α1-subunits of LTCCs contain a polybasic amino acid sequence within their I-II linkers that binds to the plasma membrane. This polybasic motif is required for normal channel gating and modulation. Conclusion: The polybasic cluster stabilizes normal channel activity. Significance: We discovered a new modulatory domain of LTCCs within their pore-forming α1-subunit. L-type voltage-gated Ca2+ channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I–IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca2+ levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca2+ channel function.

New gain-of-function mutation shows CACNA1D as recurrently mutated gene in autism spectrum disorders and epilepsy

Abstract CACNA1D encodes the pore-forming α1-subunit of Cav1.3, an L-type voltage-gated Ca2+-channel. Despite the recent discovery of two de novo missense gain-of-function mutations in Cav1.3 in two individuals with autism spectrum disorder (ASD) and intellectual disability CACNA1D has not been considered a prominent ASD-risk gene in large scale genetic analyses, since such studies primarily focus on likely-disruptive genetic variants. Here we report the discovery and characterization of a third de novo missense mutation in CACNA1D (V401L) in a patient with ASD and epilepsy. For the functional characterization we introduced mutation V401L into two major C-terminal long and short Cav1.3 splice variants, expressed wild-type or mutant channel complexes in tsA-201 cells and performed whole-cell patch-clamp recordings. Mutation V401L, localized within the channel’s activation gate, significantly enhanced current densities, shifted voltage dependence of activation and inactivation to more negative voltages and reduced channel inactivation in both Cav1.3 splice variants. Altogether, these gating changes are expected to result in enhanced Ca2+-influx through the channel, thus representing a strong gain-of-function phenotype. Additionally, we also found that mutant channels retained full sensitivity towards the clinically available Ca2+ -channel blocker isradipine. Our findings strengthen the evidence for CACNA1D as a novel candidate autism risk gene and encourage experimental therapy with available channel-blockers for this mutation. The additional presence of seizures and neurological abnormalities in our patient define a novel phenotype partially overlapping with symptoms in two individuals with PASNA (congenital primary aldosteronism, seizures and neurological abnormalities) caused by similar Cav1.3 gain-of-function mutations.

Mechanisms Responsible for ω-Pore Currents in Cav Calcium Channel Voltage-Sensing Domains

Mutations of positively charged amino acids in the S4 transmembrane segment of a voltage-gated ion channel form ion-conducting pathways through the voltage-sensing domain, named ω-current. Here, we used structure modeling and MD simulations to predict pathogenic ω-currents in CaV1.1 and CaV1.3 Ca2+ channels bearing several S4 charge mutations. Our modeling predicts that mutations of CaV1.1-R1 (R528H/G, R897S) or CaV1.1-R2 (R900S, R1239H) linked to hypokalemic periodic paralysis type 1 and of CaV1.3-R3 (R990H) identified in aldosterone-producing adenomas conducts ω-currents in resting state, but not during voltage-sensing domain activation. The mechanism responsible for the ω-current and its amplitude depend on the number of charges in S4, the position of the mutated S4 charge and countercharges, and the nature of the replacing amino acid. Functional characterization validates the modeling prediction showing that CaV1.3-R990H channels conduct ω-currents at hyperpolarizing potentials, but not upon membrane depolarization compared with wild-type channels.

Role of L-Type Ca2+ Channels in Sensory Cells

L-type Ca2+ channels play an important role in sensory cells present in the inner ear and the retina. Cav1.3 predominates in sensory cells of the inner ear (inner and outer cochlear hair cells and vestibular hair cells) and Cav1.4 in retinal neurons. Their pore-forming α1-subunits are highly homologous but functionally heterogeneous. Such variability is ensured either by differential interaction with modulatory proteins (such as Ca2+-binding proteins), differences in alternative splicing, posttranslational modification (RNA-editing) or in subunit composition. We will discuss special structural features that stabilize properties required for proper function in these cells and allow fine-tuning of Ca2+ signals. Whereas so far only one Cav1.3 human disease mutation has been published more than 50 mutations have been reported for Cav1.4. Cav1.3 channels are currently discussed as molecular target for neuroprotection in Parkinsons Disease. Are their regulatory mechanisms also interesting for potential pharmacotherapeutic modulation? How do loss- and gain-of-function mutations on the protein level both result in impaired retinal synaptic transmission in patients carrying these mutations? We will summarize our current knowledge about the role of L-type channels for human hearing and visual disorders.

Aldosterone-Producing Adenomas

Mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1 are thought to cause the excessive autonomous aldosterone secretion of aldosterone-producing adenomas (APAs). The histopathology of KCNJ5 mutant APAs, the most common and largest, has been thoroughly investigated and shown to have a zona fasciculata–like composition. This study aims to characterize the histopathologic spectrum of the other genotypes and document the proliferation rate of the different sized APAs. Adrenals from 39 primary aldosteronism patients were immunohistochemically stained for CYP11B2 to confirm diagnosis of an APA. Twenty-eight adenomas had sufficient material for further analysis and were target sequenced at hot spots in the 5 causal genes. Ten adenomas had a KCNJ5 mutation (35.7%), 7 adenomas had an ATP1A1 mutation (25%), and 4 adenomas had a CACNA1D mutation (14.3%). One novel mutation in exon 28 of CACNA1D (V1153G) was identified. The mutation caused a hyperpolarizing shift of the voltage-dependent activation and inactivation and slowed the channel’s inactivation kinetics. Immunohistochemical stainings of CYP17A1 as a zona fasciculata cell marker and Ki67 as a proliferation marker were used. KCNJ5 mutant adenomas showed a strong expression of CYP17A1, whereas ATP1A1/CACNA1D mutant adenomas had a predominantly negative expression (P value =1.20×10−4). ATP1A1/CACNA1D mutant adenomas had twice the nuclei with intense staining of Ki67 than KCNJ5 mutant adenomas (0.7% [0.5%–1.9%] versus 0.4% [0.3%–0.7%]; P value =0.04). Further, 3 adenomas with either an ATP1A1 mutation or a CACNA1D mutation had >30% nuclei with moderate Ki67 staining. In summary, similar to KCNJ5 mutant APAs, ATP1A1 and CACNA1D mutant adenomas have a seemingly specific histopathologic phenotype.

Aldosterone-Producing AdenomasNovelty and Significance: Histopathology–Genotype Correlation and Identification of a Novel CACNA1D Mutation

Mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1 are thought to cause the excessive autonomous aldosterone secretion of aldosterone-producing adenomas (APAs). The histopathology of KCNJ5 mutant APAs, the most common and largest, has been thoroughly investigated and shown to have a zona fasciculata–like composition. This study aims to characterize the histopathologic spectrum of the other genotypes and document the proliferation rate of the different sized APAs. Adrenals from 39 primary aldosteronism patients were immunohistochemically stained for CYP11B2 to confirm diagnosis of an APA. Twenty-eight adenomas had sufficient material for further analysis and were target sequenced at hot spots in the 5 causal genes. Ten adenomas had a KCNJ5 mutation (35.7%), 7 adenomas had an ATP1A1 mutation (25%), and 4 adenomas had a CACNA1D mutation (14.3%). One novel mutation in exon 28 of CACNA1D (V1153G) was identified. The mutation caused a hyperpolarizing shift of the voltage-dependent activation and inactivation and slowed the channel’s inactivation kinetics. Immunohistochemical stainings of CYP17A1 as a zona fasciculata cell marker and Ki67 as a proliferation marker were used. KCNJ5 mutant adenomas showed a strong expression of CYP17A1, whereas ATP1A1/CACNA1D mutant adenomas had a predominantly negative expression (P value =1.20×10−4). ATP1A1/CACNA1D mutant adenomas had twice the nuclei with intense staining of Ki67 than KCNJ5 mutant adenomas (0.7% [0.5%–1.9%] versus 0.4% [0.3%–0.7%]; P value =0.04). Further, 3 adenomas with either an ATP1A1 mutation or a CACNA1D mutation had >30% nuclei with moderate Ki67 staining. In summary, similar to KCNJ5 mutant APAs, ATP1A1 and CACNA1D mutant adenomas have a seemingly specific histopathologic phenotype.