Petronel Tuluc

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.

A CaV1.1 Ca2+ Channel Splice Variant with High Conductance and Voltage-Sensitivity Alters EC Coupling in Developing Skeletal Muscle

The Ca(2+) channel alpha(1S) subunit (Ca(V)1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca(2+) release from internal stores and conducts a slowly activating Ca(2+) current. However, this Ca(2+) current is not essential for skeletal muscle EC coupling. Here, we identified a Ca(V)1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged alpha(1S) subunit and expressed it in dysgenic (alpha(1S)-null) myotubes. GFP-alpha(1S)Delta 29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca(2+) currents through GFP-alpha(1S)Delta 29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca(2+) influx contributed substantially to the depolarization-induced Ca(2+) transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary alpha(2)delta-1 subunit. Thus, characterizing the Ca(V)1.1 Delta 29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca(2+) channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle.

Auxiliary Ca2+ channel subunits: lessons learned from muscle

Voltage-gated Ca(2+) channels are multi-subunit complexes involved in many key functions of excitable cells. A multitude of studies in heterologous cells demonstrated that coexpression of the pore-forming alpha(1) subunits with auxiliary alpha(2)delta and beta subunits promotes membrane expression and modulates the biophysical channel properties. New null-mutant animal models and shRNA based knockdown experiments in skeletal muscle cells for the first time demonstrated the physiological roles and possible pathological effects of the alpha(2)delta-1 and beta(1a) subunits in a differentiated excitable cell. The alpha(2)delta-1 subunit is the determinant of the typical current properties of skeletal and cardiac muscle Ca(2+) channels. The beta(1a) subunit links the skeletal muscle Ca(2+) channel to the Ca(2+) release channel in the sarcoplasmic reticulum. Whether these specific functions in muscle indicate similar roles of alpha(2)delta and beta subunits as functional modulator and structural organizer, respectively, in neurons is being discussed.

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.

Surface traffic of dendritic CaV1.2 calcium channels in hippocampal neurons.

In neurons L-type calcium currents function in gene regulation and synaptic plasticity, while excessive calcium influx leads to excitotoxicity and neurodegeneration. The major neuronal CaV1.2 L-type channels are localized in clusters in dendritic shafts and spines. Whereas CaV1.2 clusters remain stable during NMDA-induced synaptic depression, L-type calcium currents are rapidly downregulated during strong excitatory stimulation. Here we used fluorescence recovery after photobleaching (FRAP), live cell-labeling protocols, and single particle tracking (SPT) to analyze the turnover and surface traffic of CaV1.2 in dendrites of mature cultured mouse and rat hippocampal neurons, respectively. FRAP analysis of channels extracellularly tagged with superecliptic pHluorin (CaV1.2-SEP) demonstrated ∼20% recovery within 2 min without reappearance of clusters. Pulse–chase labeling showed that membrane-expressed CaV1.2-HA is not internalized within1 h, while blocking dynamin-dependent endocytosis resulted in increased cluster density after 30 min. Together, these results suggest a turnover rate of clustered CaV1.2s on the hour time scale. Direct recording of the lateral movement in the membrane using SPT demonstrated that dendritic CaV1.2s show highly confined mobility with diffusion coefficients of ∼0.005 μm2 s−1. Consistent with the mobile CaV1.2 fraction observed in FRAP, a ∼30% subpopulation of channels reversibly exchanged between confined and diffusive states. Remarkably, high potassium depolarization did not alter the recovery rates in FRAP or the diffusion coefficients in SPT analyses. Thus, an equilibrium of clustered and dynamic CaV1.2s maintains stable calcium channel complexes involved in activity-dependent cell signaling, whereas the minor mobile channel pool in mature neurons allows limited capacity for short-term adaptations.

Identification and functional characterization of malignant hyperthermia mutation T1354S in the outer pore of the Cavα1S-subunit

To identify the genetic locus responsible for malignant hyperthermia susceptibility (MHS) in an Italian family, we performed linkage analysis to recognized MHS loci. All MHS individuals showed cosegregation of informative markers close to the voltage-dependent Ca(2+) channel (Ca(V)) α(1S)-subunit gene (CACNA1S) with logarithm of odds (LOD)-score values that matched or approached the maximal possible value for this family. This is particularly interesting, because so far MHS was mapped to >178 different positions on the ryanodine receptor (RYR1) gene but only to two on CACNA1S. Sequence analysis of CACNA1S revealed a c.4060A>T transversion resulting in amino acid exchange T1354S in the IVS5-S6 extracellular pore-loop region of Ca(V)α(1S) in all MHS subjects of the family but not in 268 control subjects. To investigate the impact of mutation T1354S on the assembly and function of the excitation-contraction coupling apparatus, we expressed GFP-tagged α(1S)T1354S in dysgenic (α(1S)-null) myotubes. Whole cell patch-clamp analysis revealed that α(1S)T1354S produced significantly faster activation of L-type Ca(2+) currents upon 200-ms depolarizing test pulses compared with wild-type GFP-α(1S) (α(1S)WT). In addition, α(1S)T1354S-expressing myotubes showed a tendency to increased sensitivity for caffeine-induced Ca(2+) release and to larger action-potential-induced intracellular Ca(2+) transients under low (≤ 2 mM) caffeine concentrations compared with α(1S)WT. Thus our data suggest that an additional influx of Ca(2+) due to faster activation of the α(1S)T1354S L-type Ca(2+) current, in concert with higher caffeine sensitivity of Ca(2+) release, leads to elevated muscle contraction under pharmacological trigger, which might be sufficient to explain the MHS phenotype.

Computer modeling of siRNA knockdown effects indicates an essential role of the Ca2+ channel α2δ-1 subunit in cardiac excitation–contraction coupling

L-type Ca2+ currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca2+ signal, which regulates the contraction force. The auxiliary Ca2+ channel subunits α2δ-1 and β2 are important regulators of membrane expression and current properties of the cardiac Ca2+ channel (CaV1.2). However, their role in cardiac excitation–contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the α2δ-1 subunit in a muscle expression system with simulation of APs and Ca2+ transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with CaV1.2 (GFP-α1C) recapitulates key properties of cardiac excitation–contraction coupling. Concomitant depletion of the α2δ-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-α1C subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, α2δ-1 depletion shifted the voltage dependence of Ca2+ current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca2+ concentration during each contraction. Thus, the Ca2+ channel α2δ-1 subunit is not essential for normal Ca2+ channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of α2δ-1 function is predicted to severely perturb normal heart function.

The role of auxiliary dihydropyridine receptor subunits in muscle

The skeletal muscle dihydropyridine receptor is a slowly-activating calcium channel that functions as the voltage sensor in excitation-contraction coupling. In addition to the pore-forming α1S subunit it contains the transmembrane α2δ-1 and γ1 subunits and the cytoplasmic β1a subunit. Although the roles of the auxiliary subunits in calcium channel function have been intensively studied in heterologous expression systems, their functions in excitation-contraction coupling has only recently been elucidated in muscle cells of various null-mutant animal models. In this article we will briefly outline the current state of these investigations.

Pyrimidine-2,4,6-triones are a new class of voltage-gated L-type Ca2+ channel activators

Cav1.2 and Cav1.3 are the main L-type Ca2+ channel subtypes in the brain. Cav1.3 channels have recently been implicated in the pathogenesis of Parkinson’s disease. Therefore, Cav1.3-selective blockers are developed as promising neuroprotective drugs. We studied the pharmacological properties of a pyrimidine-2,4,6-trione derivative (1-(3-chlorophenethyl)-3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione, Cp8) recently reported as the first highly selective Cav1.3 blocker. Here we show, in contrast to this previous study, that Cp8 reproducibly increases inward Ca2+ currents of Cav1.3 and Cav1.2 channels expressed in tsA-201 cells by slowing activation, inactivation and enhancement of tail currents. Similar effects are also observed for native Cav1.3 and Cav1.2 channels in mouse chromaffin cells, while non-L-type currents are unaffected. Evidence for a weak and non-selective inhibition of Cav1.3 and Cav1.2 currents is only observed in a minority of cells using Ba2+ as charge carrier. Therefore, our data identify pyrimidine-2,4,6-triones as Ca2+ channel activators.

Stable incorporation versus dynamic exchange of β subunits in a native Ca2+ channel complex

Summary Voltage-gated Ca2+ channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation–contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary &bgr; subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether &bgr; subunits can reversibly interact with, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged &agr;1 and &bgr; subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca2+ channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle &bgr;1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle &bgr;2a and &bgr;4b isoforms dynamically interacted with both &agr;1 subunits. Neither replacing the I–II loop of CaV1.1 with that of CaV2.1, nor deletions in the proximal I–II loop, known to change the orientation of &bgr; relative to the &agr;1 subunit, altered the specific dynamic properties of the &bgr; subunits. In contrast, a single residue substitution in the &agr; interaction pocket of &bgr;1aM293A increased the FRAP rate threefold. Taken together, these findings indicate that in skeletal muscle triads the homologous &bgr;1a subunit forms a stable complex, whereas the heterologous &bgr;2a and &bgr;4b subunits form dynamic complexes with the Ca2+ channel. The distinct binding properties are not determined by differences in the I–II loop sequences of the &agr;1 subunits, but are intrinsic properties of the &bgr; subunit isoforms.