Andreas Lieb

Loss of Ca v 1.3 ( CACNA1D ) function in a human channelopathy with bradycardia and congenital deafness

Deafness is genetically very heterogeneous and forms part of several syndromes. So far, delayed rectifier potassium channels have been linked to human deafness associated with prolongation of the QT interval on electrocardiograms and ventricular arrhythmia in Jervell and Lange-Nielsen syndrome. Cav1.3 voltage-gated L-type calcium channels (LTCCs) translate sound-induced depolarization into neurotransmitter release in auditory hair cells and control diastolic depolarization in the mouse sinoatrial node (SAN). Human deafness has not previously been linked to defects in LTCCs. We used positional cloning to identify a mutation in CACNA1D, which encodes the pore-forming α1 subunit of Cav1.3 LTCCs, in two consanguineous families with deafness. All deaf subjects showed pronounced SAN dysfunction at rest. The insertion of a glycine residue in a highly conserved, alternatively spliced region near the channel pore resulted in nonconducting calcium channels that had abnormal voltage-dependent gating. We describe a human channelopathy (termed SANDD syndrome, sinoatrial node dysfunction and deafness) with a cardiac and auditory phenotype that closely resembles that of Cacna1d−/− mice.

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.

The coumarin scopoletin potentiates acetylcholine release from synaptosomes, amplifies hippocampal long-term potentiation and ameliorates anticholinergic- and age-impaired memory.

In a previous study the simple, naturally derived coumarin scopoletin (SCT) was identified as an inhibitor of acetylcholinesterase (AChE), using a pharmacophore-based virtual screening approach. In this study the potential of SCT as procholinergic and cognition-enhancing therapeutic was investigated in a more detailed way, using different experimental approaches like measuring newly synthesized acetylcholine (ACh) in synaptosomes, long-term potentiation (LTP) experiments in hippocampal slices, and behavior studies. SCT enhanced the K+-stimulated release of ACh from rat frontal cortex synaptosomes, showing a bell-shaped dose effect curve (Emax: 4 μM). This effect was blocked by the nicotinic ACh receptor (nAChR) antagonists mecamylamine (MEC) and dihydro-β-erythroidine (DHE). The nAChR agonist (and AChE inhibitor) galantamine induced a similar increase in ACh release (Emax: 1 μM). SCT potentiated LTP in hippocampal slices of rat brain. The high-frequency stimulation (HFS)-induced, N-methyl-D-aspartate (NMDA) receptor dependent LTP of field excitatory postsynaptic potentials at CA3-CA1 synapses was greatly enhanced by pre-HFS application of SCT (4 μM for 4 min). This effect was mimicked by nicotine (2 μM) and abolished by MEC, suggesting an effect on nAChRs. SCT did not restore the total inhibition of LTP by NMDA receptor antagonist d, l-2-amino-5-phosphonopentanoic acid (AP-5). SCT (2 μg, i.c.v.) increased T-maze alternation and ameliorated novel object recognition of mice with scopolamine-induced cholinergic deficit. It also reduced age-associated deficits in object memory of 15–18-month-old mice (2 mg/kg sc). Our findings suggest that SCT possesses memory-improving properties, which are based on its direct nAChR agonistic activity. Therefore, SCT might be able to rescue impaired cholinergic functions by enhancing nAChR-mediated release of neurotransmitters and promoting neural plasticity in hippocampus.

C-Terminal Modulatory Domain Controls Coupling of Voltage-Sensing to Pore Opening in Cav1.3 L-type Ca2+ Channels

Activity of voltage-gated Cav1.3 L-type Ca2+ channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Cav1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (QON-V) and of current activation (ICa-V) of the long (Cav1.3L) and a short Cav1.3 splice variant (Cav1.342A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (QON) of Cav1.3L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Cav1.2 and Cav3.1. However, a significantly higher fraction of the total charge had to move for activation of Cav1.3 half-maximal conductance (Cav1.3: 68%; Cav1.2: 52%; Cav3.1: 22%). This indicated a weaker coupling of Cav1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Cav1.342A, thereby shifting ICa-V by 7.2 mV to potentials that were more negative without changing QON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of QON-V and a more negative activation of ICa-V of all three channels. These findings illustrate that the voltage sensors of Cav1.3 channels respond more sensitively to depolarization than those of Cav1.2 or Cav3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Cav1.342A splice variants to activate at lower voltages without affecting QON-V.

Effects of the coumarin scopoletin on learning and memory, on release of acetylcholine from brain synaptosomes and on long-term potentiation in hippocampus

Background Among the chemical class of coumarins several substances have been described to be effective as cognition enhancers. We have recently characterized the coumarin scopoletin as a compound which fits a pharmacophore model of AChE inhibitors and enhances the release of ACh in rat brain [1]. Now, a comprehensive study was carried out in order to investigate the effects of scopoletin on learning and memory, on release of ACh from synaptosomes and on hippocampal long-term potentiation (LTP) in order to uncover the mechanism of action.

Cell-type-specific tuning of Cav1.3 Ca2+-channels by a C-terminal automodulatory domain

Cav1.3 L-type Ca2+-channel function is regulated by a C-terminal automodulatory domain (CTM). It affects channel binding of calmodulin and thereby tunes channel activity by interfering with Ca2+- and voltage-dependent gating. Alternative splicing generates short C-terminal channel variants lacking the CTM resulting in enhanced Ca2+-dependent inactivation and stronger voltage-sensitivity upon heterologous expression. However, the role of this modulatory domain for channel function in its native environment is unkown. To determine its functional significance in vivo, we interrupted the CTM with a hemagglutinin tag in mutant mice (Cav1.3DCRDHA/HA). Using these mice we provide biochemical evidence for the existence of long (CTM-containing) and short (CTM-deficient) Cav1.3 α1-subunits in brain. The long (HA-labeled) Cav1.3 isoform was present in all ribbon synapses of cochlear inner hair cells. CTM-elimination impaired Ca2+-dependent inactivation of Ca2+-currents in hair cells but increased it in chromaffin cells, resulting in hyperpolarized resting potentials and reduced pacemaking. CTM disruption did not affect hearing thresholds. We show that the modulatory function of the CTM is affected by its native environment in different cells and thus occurs in a cell-type specific manner in vivo. It stabilizes gating properties of Cav1.3 channels required for normal electrical excitability.

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.

Biophysical Properties of a Human Disease-Causing Mutation in CaV1.3 L-Type Calcium Channels

Mice lacking functional Cav1.3 L-type Ca2+-channels show sinoatrial node dysfunction and are congenitally deaf. Here we investigated the functional consequences of a homozygote mutation (insertion of an amino acid residue in a pore-forming S6-helix) in the alpha1-subunit encoding the CACNA1D gene which causes bradycardia and deafness in humans.We expressed human wildtype (WT) and mutant channel complexes (MUT) in tsA-201 cells (with alpha2delta and beta3-subunits) and recorded ON-gating (Qon) and ionic Ca2+-currents (ICa) using the whole-cell patch-clamp technique. Full length WT and MUT channel alpha1 subunit proteins were expressed at equal levels in tsA-201 membranes. In contrast to WT, MUT channels did not conduct significant ICa, but gave clear rise to Qon. WT and MUT Qon exhibited a typical nonlinear voltage-dependence of activation. Under identical experimental conditions (block of ICa by replacing Ca2+ with Mg2+ and addition of La3+ and Cd2+ in the recording solution) the half maximal activation voltage (Vh) of MUT Qon was significantly shifted by 16.2±2.8 mV (n=6-8, p<0.0001) to more negative voltages compared to WT. Inward ICa of WT activated 11.5±2.9 mV more positive than WT Qon (n=7-8; p=0.0016). In addition, MUT Qon kinetics were significantly faster than for WT evident e.g. as shorter time constants for gating current decay over a large voltage range (−10 to +50 mV).The presence of charge movement in the absence of ionic currents implies that voltage-sensors in MUT channels move, but either fail to trigger pore opening or prevent conduction through opened channels. The mutation is not located within voltage-sensing S4-helices, but within a region of S6 predicted to interact with the voltage-sensor. Compromising this functional module may uncouple voltage-sensor function from pore opening and allow voltage-sensor movements to occur faster and at more negative voltages.

Molecular Basis of a C Terminal Modulatory Mechanism in Cav1.3 Voltage-Gated Ca2+ Channels

We have previously discovered an intramolecular interaction between proximal- (PCRD) and distal C-terminal (DCRD) modulatory domains in human Cav1.3 L-type Ca2+ channels (LTCCs) which affects channel activation and inactivation gating properties (Singh et al 2008). This is present in the long (hCav1.342) but not a short (hCav1.342A) splice variant. Interestingly, this regulation has not been reported for rat Cav1.3 channel analogues (Xu and Lipscombe 2001). We systematically compared the functional properties of long and short Cav1.3 splice variants of mouse and rat with human channels after expression in tsA-201 cells using the whole-cell patch-clamp technique. The C-terminal modulation was also present in mouse (V0.5act[mV]: mCav1.342 −4.1±0.4 n=36; mCav1.342A −12.0±0.5 n=25; p<0.0001, Mann-Whitney-test) and indistinguishable from human (V0.5act[mV]: hCav1.342 −3.9±0.6 n=33; hCav1.342A −11.2±0.7 n=12; p<0.0001, Mann-Whitney-test) but was absent in rat. Exon 11, which is only present in rat Cav1.3, is not responsible for the difference suggesting single amino acid exchanges in other transmembrane domains or within the N- or C-terminus to account for the species difference. Additionally, we report a new short Cav1.3 splice variant (hCav1.343S) identified in human and mouse brain tissue. The voltage-dependence of hCav1.343SICa activation and inactivation was significantly shifted to more hyperpolarized potentials (V0.5act[mV]: hCav1.3S: −12.4±1.0, n=10, p<0.0001; V0.5inact[mV]: hCav1.342: −2.7±0.6, n=12, p<0.0001, Mann-Whitney-test) and channel inactivation was significantly faster compared to the long form (% inactivation during 250ms at Vmax: Cav1.342: 63.6±2.4; Cav1.343S: 87.0±1.5; p<0.0001, Mann-Whitney-test). These gating differences are due to the lack of the DCRD. In contrast to hCav1.342A, hCav1.343S still contains the PCRD. We therefore hypothesize that this short form can be modulated by binding the DCRD domain of adjacent LTCCs or the free C-terminal peptide derived from Cav1.2. Support: FWF (P-20670, JS), University of Innsbruck (AK).