Mathias Gebhart

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.

C-terminal modulator controls Ca2+-dependent gating of Cav1.4 L-type Ca2+ channels

Tonic neurotransmitter release at sensory cell ribbon synapses is mediated by calcium (Ca2+) influx through L-type voltage-gated Ca2+ channels. This tonic release requires the channels to inactivate slower than in other tissues. Cav1.4 L-type voltage-gated Ca2+ channels (LTCCs) are found at high densities in photoreceptor terminals, and α1 subunit mutations cause human congenital stationary night blindness type-2 (CSNB2). Cav1.4 voltage-dependent inactivation is slow and Ca2+-dependent inactivation (CDI) is absent. We show that removal of the last 55 or 122 (C122) C-terminal amino acid residues of the human α1 subunit restores calmodulin-dependent CDI and shifts voltage of half-maximal activation to more negative potentials. The C terminus must therefore form part of a mechanism that prevents calmodulin-dependent CDI of Cav1.4 and controls voltage-dependent activation. Fluorescence resonance energy transfer experiments in living cells revealed binding of C122 to C-terminal motifs mediating CDI in other Ca2+ channels. The absence of this modulatory mechanism in the CSNB2 truncation mutant K1591X underlines its importance for normal retinal function in humans.

Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.

Low voltage activation of CaV1.3 L-type Ca2+ channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. CaV1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in CaV1.4 L-type Ca2+ channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the CaV1.3 α1 subunit C terminus gives rise to a long (CaV1.342) and a short form (CaV1.342A), we investigated if a C-terminal modulatory mechanism also controls CaV1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with β3 and α2δ1 subunits in HEK-293 cells. Activation of calcium current through CaV1.342A channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several CaV1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting CaV1.3ΔC116 channels showed gating properties similar to CaV1.342A that were reverted by co-expression of the corresponding C-terminal peptide C116. Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of CaV1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control CaV1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates CaV1.3 channel activation at lower voltages expected to favor CaV1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.

Functional Properties of a Newly Identified C-terminal Splice Variant of Cav1.3 L-type Ca2+ Channels

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Cav1.3 L-type Ca2+ channels (Cav1.3L) is a major determinant of their voltage- and Ca2+-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Cav1.342A channels that activate at a more negative voltage range and exhibit more pronounced Ca2+-dependent inactivation. Here we describe the discovery of a novel short splice variant (Cav1.343S) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Cav1.342A, still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Cav1.343S also activated at more negative voltages like Cav1.342A but Ca2+-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Cav1.3L. The presence of the proximal C terminus in Cav1.343S channels preserved their modulation by distal C terminus-containing Cav1.3- and Cav1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca2+ influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Cav1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca2+ channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca2+ accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca2+-induced neurodegenerative processes.

Modulation of Cav1.3 Ca2+ channel gating by Rab3 interacting molecule

Neurotransmitter release and spontaneous action potentials during cochlear inner hair cell (IHC) development depend on the activity of Ca(v)1.3 voltage-gated L-type Ca(2+) channels. Their voltage- and Ca(2+)-dependent inactivation kinetics are slower than in other tissues but the underlying molecular mechanisms are not yet understood. We found that Rab3-interacting molecule-2alpha (RIM2alpha) mRNA is expressed in immature cochlear IHCs and the protein co-localizes with Ca(v)1.3 in the same presynaptic compartment of IHCs. Expression of RIM proteins in tsA-201 cells revealed binding to the beta-subunit of the channel complex and RIM-induced slowing of both Ca(2+)- and voltage-dependent inactivation of Ca(v)1.3 channels. By inhibiting inactivation, RIM induced a non-inactivating current component typical for IHC Ca(v)1.3 currents which should allow these channels to carry a substantial window current during prolonged depolarizations. These data suggest that RIM2 contributes to the stabilization of Ca(v)1.3 gating kinetics in immature IHCs.

Reciprocal Interactions Regulate Targeting of Calcium Channel β Subunits and Membrane Expression of α1 Subunits in Cultured Hippocampal Neurons

Auxiliary β subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca2+ channels in heterologous cells. In brain, all four β isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of β subunits and their role in membrane expression of CaV1.2 α1 subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous β subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged β subunits demonstrated that all β subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic CaV1.2, thus indicating a great promiscuity in α1-β interactions. In contrast, restricted axonal targeting of β1 and weak colocalization of β4b with CaV1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged CaV1.2 was strongly enhanced by all β subunits in an isoform-specific manner. Conversely, mutating the α-interaction domain of CaV1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a β subunit with the α-interaction domain is absolutely essential for membrane expression of α1 subunits, as well as for the subcellular localization of β subunits, which by themselves possess little or no targeting properties.

Activity and calcium regulate nuclear targeting of the calcium channel beta4b subunit in nerve and muscle cells

Auxiliary beta subunits are critical determinants of membrane expression and gating properties of voltage-gated calcium channels. Mutations in the beta4 subunit gene cause ataxia and epilepsy. However, the specific function of beta4 in neurons and its causal relation to neurological diseases are unknown. Here we report the localization of the beta4 subunit in the nuclei of cerebellar granule and Purkinje cells. beta4b was the only beta isoform showing nuclear targeting when expressed in neurons and skeletal myotubes. Its specific nuclear targeting property was mapped to an N-terminal double-arginine motif, which was necessary and sufficient for targeting beta subunits into the nucleus. Spontaneous electrical activity and calcium influx negatively regulated beta4b nuclear localization by a CRM-1-dependent nuclear export mechanism. The activity-dependent shuttling of beta4b into and out of the nucleus indicates a specific role of this beta subunit in neurons, in communicating the activity of calcium channels to the nucleus.

Structural determinants of CaV1.3 L-type calcium channel gating

channels, in particular the voltage dependence of activation (V 0.5 ) and Ca 2+ dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Ca v 1.3 (Ca v 1.3 L ), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCa v 1.3 scg ). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCa v 1.3 scg with a rat cDNA isolated from rat pancreas (rCa v 1.3 L ). When expressed in tsA-201 cells under identical experimental conditions rCa v 1.3 L exhibited Ca 2+ current properties indistinguishable from human and mouse Ca v 1.3 L , compatible with the presence of a functional CTM. In contrast, rCa v 1.3 scg showed gating properties similar to human short splice variants lacking a CTM. rCa v 1.3 scg differs from rCa v 1.3 L at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-terminal polymethionine stretch with two additional lysines. Two aa (S244, A2075) in rCa v 1.3 scg explained most of the functional differences to rCa v 1.3 L . Their mutation to the corresponding residues in rCa v 1.3 L (G244, V2075) revealed that both contributed to the more negative V 0.5 , but caused opposite effects on CDI. A2075 (located within a region forming the CTM) additionally permitted higher channel open probability. The cooperative action in the double-mutant restored gating properties similar to rCa v 1.3 L . We found no evidence for transcripts containing one of the single rCa v 1.3 scg mutations in rat superior cervical ganglion preparations. However, the rCa v 1.3 scg variant provided interesting insight into the structural machinery involved in Ca v 1.3 gating.

A mouse model to study the C-terminal regulation of CaV1.3 L-type calcium channels

Background CaV1.3 voltage-gated L-type Ca 2+ channels (LTCCs) play an important role for hearing, cardiac pacemaking and neuronal excitability. The C-terminus of CaV1.3 tightly controls channel gating by an intramolecular protein interaction involving two putative a-helices (termed PCRD, DCRD), which form a C-terminal modulatory mechanism (CTM) only in full-length CaV1.3 variants. In short (CaV1.342A and CaV1.343S) CaV1.3 a1 subunit splice variants CTM is absent which leads to profound changes in channel gating: activation occurs at more negative voltages and Ca-dependent inactivation (CDI) is faster.

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.