L-type Calcium Channel Cav1 Research Articles

Calcium/calmodulin-dependent serine protein kinase CASK modulates the L-type calcium current

AimThe L-type voltage-gated calcium channel Cav1.2 mediates the calcium influx into cells upon membrane depolarization. The list of cardiopathies associated to Cav1.2 dysfunctions highlights the importance of this channel in cardiac physiology. Calcium/calmodulin-dependent serine protein kinase (CASK), expressed in cardiac cells, has been identified as a regulator of Cav2.2 channels in neurons, but no experiments have been performed to investigate its role in Cav1.2 regulation. Methods and resultsFull length or the distal C-terminal truncated of the pore-forming Cav1.2 channel (Cav1.2α1c), both present in cardiac cells, were expressed in TsA-201 cells. In addition, a shRNA silencer, or scramble as negative control, of CASK was co-transfected in order to silence CASK endogenously expressed. Three days post-transfection, the barium current was increased only for the truncated form without alteration of the steady state activation and inactivation biophysical properties. The calcium current, however, was increased after CASK silencing with both types of Cav1.2α1c subunits suggesting that, in absence of calcium, the distal C-terminal counteracts the CASK effect. Biochemistry experiments did not reveals neither an alteration of Cav1.2 channel protein expression after CASK silencing nor an interaction between Cav1.2α1c subunits and CASK. Nevertheless, after CASK silencing, single calcium channel recordings have shown an increase of the voltage-gated calcium channel Cav1.2 open probability explaining the increase of the whole-cell current. ConclusionThis study suggests CASK as a novel regulator of Cav1.2 via a modulation of the voltage-gated calcium channel Cav1.2 open probability.

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Abstract 134: Increased Ca2+ Influx Through Cav1.2 in Vascular Smooth Muscle Cells Causes Hypertension Due to Impairment of Acetylcholine-dependent Vascular Relaxation

Calcium channel plays an important role in smooth muscle contraction and relaxation and L-type calcium channel (Cav1.2) antagonist is widely used for treating hypertension. However, whether increasing Ca2+ influx into smooth muscle cells results in hypertension (through increasing smooth muscle cell contractility) or hypotension (through activating BKCa channels) is unclear. Aiming to answer this question, we established a transgenic (TG) mouse model with smooth muscle cell specific (SM22-α promoter driven) overexpression of a splicing variant of Cavβ2 subunit, Cavβ2a, tagged with GFP. Methods and Results: Immunostaining showed that Cavβ2a-GFP expressed in smooth muscle cells specifically and L-type Ca2+ current in smooth muscle cells from the 4 th branch of mesenteric arteries of transgenic mice was significantly increased (5.1±0.7pA/pF in TG, n=12 vs. 2.2±0.5pA/pF in control, n=10). Telemetric blood pressure measurement showed that 24h-averaged systolic pressure was significantly increased (135.2±9.8mmHg, n=25) compared to that of that of control c57bl/6 mice (110.2±7.8mmHg, n=16). The diastolic pressure was also significantly elevated in TG mice than in control mice. Maximum contractility of the 4 th branch mesenteric arteries measured with 40mM KCl was not different between control and TG groups. Sodium nitroprusside relaxed the phenylephrine-precontracted mesenteric artery in the same dose-dependent manner However, acetylcholine induced mesenteric artery relaxation was signficantly impaired in TG mesenteric arteries. Conclusions: Increasing Ca2+ influx through the L-type Ca2+ channel in smooth muscle cells causing hypertension via impairment of acetylcholine-dependent relaxation.

A novel mouse model of the aged brain: Over-expression of the L-type voltage-gated calcium channel CaV1.3

The aged population is growing rapidly, which has sparked tremendous interest in elucidating mechanisms of aging in both the body and the brain. Animal models have become an indispensable tool in biomedical science, but because of the cost and extended timeframe associated with aging animals to appropriate time points, studies that rely on using aged animals are often not feasible. Somewhat surprisingly, there are relatively few animal models that have been specifically engineered to mimic physiological changes known to occur during “normal” aging. Developing transgenic animal models that faithfully mimic key aspects of aging would likely be of great utility in studying both age-related deficits in the absence of overt pathology as well as an adjunct for transgenic models of diseases where aging is a primary risk factor.In particular, there are several alterations in the aged brain that are amenable to being modeled genetically. We have focused on one key aspect that has been repeatedly demonstrated in aged animals – an increase in the L-type voltage-gated calcium channel CaV1.3. Here we present a novel transgenic mouse line in which expression of CaV1.3 is increased by approximately 50% in the forebrain of young mice. These mice do not display any overt physical or non-cognitive deficits, exhibiting normal exploratory behavior, motor function, and affective-like responses, suggesting that these mice can be successfully deployed to assess the impact of an “aged brain” in a variety of conditions.

Regulation of aldosterone secretion by Cav1.3.

Aldosterone-producing adenomas (APAs) vary in phenotype and genotype. Zona glomerulosa (ZG)-like APAs frequently have mutations of an L-type calcium channel (LTCC) CaV1.3. Using a novel antagonist of CaV1.3, compound 8, we investigated the role of CaV1.3 on steroidogenesis in the human adrenocortical cell line, H295R, and in primary human adrenal cells. This investigational drug was compared with the common antihypertensive drug nifedipine, which has 4.5-fold selectivity for the vascular LTCC, CaV1.2, over CaV1.3. In H295R cells transfected with wild-type or mutant CaV1.3 channels, the latter produced more aldosterone than wild-type, which was ameliorated by 100 μM of compound 8. In primary adrenal and non-transfected H295R cells, compound 8 decreased aldosterone production similar to high concentration of nifedipine (100 μM). Selective CaV1.3 blockade may offer a novel way of treating primary hyperaldosteronism, which avoids the vascular side effects of CaV1.2-blockade, and provides targeted treatment for ZG-like APAs with mutations of CaV1.3.

Calcium Current Properties and Leak Conductance in Mouse Muscle Fibers Overexpressing a Type 1 Hypokalemic Periodic Paralysis Mutant L-Type Calcium Channel

Missense mutations in the gene encoding the alpha1 subunit of the L-type calcium channel CaV1.1 induce type 1 Hypokalemic Periodic Paralysis (HypoPP1). These mutations mainly occur at arginine residues in the fourth transmembrane segment of voltage-sensor domains. Very few studies have investigated the acute effects of these mutations on channel function and muscle membrane electrical properties because of the difficulty to express Cav1.1 in heterologous systems. In the present study we successfully transferred by electroporation the genes encoding the turboGFP-tagged human wildtype (WT) and R1239H HypoPP1 mutant Cav1.1 into hind limb mouse muscles. The expression profile of the two channels showed a regular striated pattern indicative of the localization of the channels in the t-tubule membrane. Measurement of the L-type current using the silicone-clamp technique showed that the maximal conductance and the voltage-dependence of the Cav1.1 channel were significantly reduced and shifted towards negative potentials respectively in fibers expressing R1239H Cav1.1 as compared to fibers expressing WT Cav1.1. Applying voltage ramps from a holding potential of 0, −20, −40 or −60 mV to −120 mV in the presence of an external low-chloride, sodium-free and potassium-free solution revealed a significant higher leak conductance measured between −80 and −120 mV in fibers expressing R1239H Cav1.1. Acidification of the external solution significantly increased the leak inward current, leak conductance and the fluorescence of an internally loaded pH indicator in fibers expressing R1239H. These data suggest that an elevated leak inward current, likely carrying protons, flows at resting membrane potentials in fibers expressing R1239H Cav1.1 and that acidification of the external medium following muscle exercise could contribute to favor the onset of muscle paralysis in HypoPP1.

T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels.

The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling.

Binding mechanisms of 1,4-dihydropyridine derivatives to L-type calcium channel Cav1.2: a molecular modeling study.

L-type Ca(2+) channels (LTCCs), the heteromultimeric proteins, are associated with electrical signaling and provide the key link between electrical signals and non-electrical processes. 1,4-Dihydropyridine (DHP) derivatives are a major class of blockers for LTCCs, and have experienced widespread use in the treatment of cardiovascular diseases. However, the precise knowledge of the binding mechanism of these ligands to LTCCs at the atomic level has remained unknown because of the unavailability of the crystal structures of LTCCs. In this study, homology modeling, molecular docking, molecular dynamics (MD) simulations, free energy calculations and decomposition were employed to explore the structural requirement of the binding of DHP derivatives to human Cav1.2, a member of LTCCs. The binding conformations of the DHPs in the active site of Cav1.2 were predicted, and the rank of the binding free energies of Cav1.2/DHPs is generally consistent with the experimental data. The structural analysis shows that most studied ligands fit into a hydrophobic pocket formed by Phe1129, Ile1173, Phe1176, Met1177 and Met1509, and form aryl-aryl interaction with Phe1129 or Tyr1508. The consistency between the predictions and experimental data suggest that the developed model is reliable and can be used as a valuable platform for the structure-based design of new potent ligands of Cav1.2.

Three-Dimensional Architecture of the L-Type Calcium Channel: Structural Insights into the CaVα2δ1 Auxiliary Protein

L-type calcium channels (LTCC) are responsible for Ca2+ influx into muscle and neurons. These macromolecular complexes minimally comprise the main poreforming CaVα1 and auxiliary subunits CaVβ and CaVα2δ1. The ultrastructure of the oligomeric LTCC complexes from heart and skeletal muscle has been reported previously at ≈ 20 A, a resolution that prevent identification of structural domains. Recent improvements in cryo-electronic microscopy (EM) methods made it possible to obtain a three-dimensional structure of the rabbit skeletal muscle LTCC CaV1.1 complex at a resolution of 4.2 A and recently at 3.6 A. This technique requires only nanograms of purified proteins and circumvents crystallization as a means for structure determination. The high resolution cryo-EM structure shows the molecular architecture of the subunits comprising the oligomeric complex and for the first time, a high-resolution glance of the largely extracellular CaVα2δ1 protein with its extracellular domains (Cache1, VWA, and Cache2). Although the CaVα2δ1 protein is a single-pass transmembrane protein, the complex topology of its extracellular domain represents a technical challenge for structure determination using conventional purification approaches. Herein we show the merits of a strategy based upon the purification of small structural domains that can be elucidated individually before these domains are reassembled into the quaternary structure. A structural model was derived using ab initio structure prediction constrained by small angle X-ray scattering profile of the refolded Cache2 domain. The excellent agreement between the predicted structure and the available cryo-EM structure suggests a novel and rapid procedure to discover structural information of protein domains.

The L-type calcium channel Cav1.3 is required for proper hippocampal neurogenesis and cognitive functions

L-type voltage gated Ca2+ channels (LTCCs) are widely expressed within different brain regions including the hippocampus. The isoforms Cav1.2 and Cav1.3 have been shown to be involved in hippocampus-dependent learning and memory, cognitive functions that require proper hippocampal neurogenesis. In vitro, functional LTCCs are expressed on neuronal progenitor cells, where they promote neuronal differentiation. Expression of LTCCs on neural stem and progenitor cells within the neurogenic regions in the adult brain in vivo has not been examined so far, and a contribution of the individual isoforms Cav1.2 and Cav1.3 to adult neurogenesis remained to be clarified. To reveal the role of these channels we first evaluated the expression patterns of Cav1.2 and Cav1.3 in the hippocampal dentate gyrus and the subventricular zone (SVZ) in adult (2- and 3-month old) and middle-aged (15-month old) mice on mRNA and protein levels. We performed immunohistological analysis of hippocampal neurogenesis in adult and middle-aged Cav1.3−/− mice and finally addressed the importance of Cav1.3 for hippocampal function by evaluating spatial memory and depression-like behavior in adult Cav1.3−/− mice. Our results showed Cav1.2 and Cav1.3 expression at different stages of neuronal differentiation. While Cav1.2 was primarily restricted to mature NeuN+ granular neurons, Cav1.3 was expressed in Nestin+ neural stem cells and in mature NeuN+ granular neurons. Adult and middle-aged Cav1.3−/− mice showed severe impairments in dentate gyrus neurogenesis, with significantly smaller dentate gyrus volume, reduced survival of newly generated cells, and reduced neuronal differentiation. Further, Cav1.3−/− mice showed impairment in the hippocampus dependent object location memory test, implicating Cav1.3 as an essential element for hippocampus-associated cognitive functions. Thus, modulation of LTCC activities may have a crucial impact on neurogenic responses and cognition, which should be considered for future therapeutic administration of LTCCs modulators.

Heterogeneity of Calcium Channel/cAMP-Dependent Transcriptional Activation.

The major function of the voltage-gated calcium channels is to provide the Ca(2+) flux into the cell. L-type voltage-gated calcium channels (Cav1) serve as voltage sensors that couple membrane depolarization to many intracellular processes. Electrical activity in excitable cells affects gene expression through signaling pathways involved in the excitation-transcription (E-T) coupling. E-T coupling starts with activation of the Cav1 channel and results in initiation of the cAMP-response element binding protein (CREB)-dependent transcription. In this review we discuss the new quantitative approaches to measuring E-T signaling events. We describe the use of wavelet transform to detect heterogeneity of transcriptional activation in nuclei. Furthermore, we discuss the properties of discovered microdomains of nuclear signaling associated with the E-T coupling and the basis of the frequency-dependent transcriptional regulation.

L-type Calcium Channel Cav1.2 Is Required for Maintenance of Auditory Brainstem Nuclei

Cav1.2 and Cav1.3 are the major L-type voltage-gated Ca(2+) channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Cav1.3 is essential for proper maturation. Here, we investigated the role of Cav1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Cav1.3, loss of Cav1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Cav1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Cav1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Cav1.2 and Cav1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue.

Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10d, 3mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change>1.5, p<0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects.

Cav1.3 Channels as Key Regulators of Neuron-Like Firings and Catecholamine Release in Chromaffin Cells.

Neuronal and neuroendocrine L-type calcium channels (Cav1.2, Cav1.3) open readily at relatively low membrane potentials and allow Ca2+ to enter the cells near resting potentials. In this way, Cav1.2 and Cav1.3 shape the action potential waveform, contribute to gene expression, synaptic plasticity, neuronal differentiation, hormone secretion and pacemaker activity. In the chromaffin cells (CCs) of the adrenal medulla, Cav1.3 is highly expressed and is shown to support most of the pacemaking current that sustains action potential (AP) firings and part of the catecholamine secretion. Cav1.3 forms Ca2+-nanodomains with the fast inactivating BK channels and drives the resting SK currents. These latter set the inter-spike interval duration between consecutive spikes during spontaneous firing and the rate of spike adaptation during sustained depolarizations. Cav1.3 plays also a primary role in the switch from “tonic” to “burst” firing that occurs in mouse CCs when either the availability of voltage-gated Na channels (Nav) is reduced or the β2 subunit featuring the fast inactivating BK channels is deleted. Here, we discuss the functional role of these “neuron-like” firing modes in CCs and how Cav1.3 contributes to them. The open issue is to understand how these novel firing patterns are adapted to regulate the quantity of circulating catecholamines during resting condition or in response to acute and chronic stress.

Regulation of Postsynaptic Stability by the L-type Calcium Channel CaV1.3 and its Interaction with PDZ Proteins.

Alterations in dendritic spine morphology and postsynaptic structure are a hallmark of neurological disorders. Particularly spine pruning of striatal medium spiny neurons and aberrant rewiring of corticostriatal synapses have been associated with the pathology of Parkinson’s disease and L-DOPA induced dyskinesia, respectively. Owing to its low activation threshold the neuronal L-type calcium channel CaV1.3 is particularly critical in the control of neuronal excitability and thus in the calcium-dependent regulation of neuronal functions. CaV1.3 channels are located in dendritic spines and contain a C-terminal class 1 PDZ domain-binding sequence. Until today the postsynaptic PDZ domain proteins shank, densin-180, and erbin have been shown to interact with CaV1.3 channels and to modulate their current properties. Interestingly experimental evidence suggests an involvement of all three PDZ proteins as well as CaV1.3 itself in regulating dendritic and postsynaptic morphology. Here we briefly review the importance of CaV1.3 and its proposed interactions with PDZ proteins for the stability of dendritic spines. With a special focus on the pathology associated with Parkinson’s disease, we discuss the hypothesis that CaV1.3 L-type calcium channels may be critical modulators of dendritic spine stability.

Chronic hypoxia in pregnancy affected vascular tone of renal interlobar arteries in the offspring.

Hypoxia during pregnancy could affect development of fetuses as well as cardiovascular systems in the offspring. This study was the first to demonstrate the influence and related mechanisms of prenatal hypoxia (PH) on renal interlobar arteries (RIA) in the 5-month-old male rat offspring. Following chronic hypoxia during pregnancy, phenylephrine induced significantly higher pressor responses and greater vasoconstrictions in the offspring. Nitric oxide mediated vessel relaxation was altered in the RIA. Phenylephrine-stimulated free intracellular calcium was significantly higher in the RIA of the PH group. The activity and expression of L-type calcium channel (Cav1.2), not T-type calcium channel (Cav3.2), was up-regulated. The whole-cell currents of calcium channels and the currents of Cav1.2 were increased compared with the control. In addition, the whole-cell K+ currents were decreased in the offspring exposed to prenatal hypoxia. Activity of large-conductance Ca2+-activated K+ channels and the expression of MaxiKα was decreased in the PH group. The results provide new information regarding the influence of prenatal hypoxia on the development of the renal vascular system, and possible underlying cellular and ion channel mechanisms involved.

Methamphetamine Alters Calcium Channel Gene DNA Methylation and Gene Expression in the Murine Heart

Methamphetamine (METH) is a widely abused illicit drug that is highly addictive and affects cardiac metabolism and contraction. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10d, 3mg/kg/d, subcutaneously) in C57Bl/6 mice. Parallel DNA methylation changes were identified using tiled DNA microarrays to implicate epigenetic reprogramming and highlight long-term cardiac impact of METH. Gene expression arrays identified 485 genes that were differentially expressed following METH (expression fold change>1.5, p<0.05). Using pathway enrichment analysis, families relating to calcium signaling and contractility were altered in the LV by METH. With DNA methylation analysis, 10.6% of probed regions of LV DNA exhibited significant changes following METH exposure; nearly 75% of those changes were hypomethylation. By comparing gene expression and DNA methylation changes, 27 genes revealed differences by both methods of analysis. Among these, only the promoter for CACNA1C, the gene encoding L-type calcium channel Cav1.2, was hypomethylated by METH, and CACNA1C gene expression was increased by METH on microarray. For confirmation, quantitative PCR verified Cav1.2 LV mRNA increased due to METH. Correlative immunoblots revealed a 3.5-fold increase in Cav1.2 protein abundance in METH LVs compared to LVs of vehicle controls. Histopathologically, contraction band necrosis was abundant in METH LVs. These results implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH and suggest possible mechanisms for METH cardiac toxicity in humans.

Super-Resolution Analysis of the Distribution of RyR, Cav1.2 and NCX within the Mammalian Couplon

We have developed a dynamically stable 3D dSTORM system, modified to give highly accurate blink positioning deep within a thick sample, to investigate the distribution and localization of the ryanodine receptor (RyR), the L-type calcium channel (Cav1.2) and the sodium-calcium exchanger (NCX) both on the surface and in the interior of the rat cardiomyocyte. The images have a resolution of 10nm in X and Y and 40 nm in Z, and cover areas of up to 1000 µm2 in XY with depths of up to 700nm in Z.We have been able to identify the position of individual RyR tetramers and have confirmed the recently published tomographic finding that in the resting state most RyR clusters are neither homogenous nor well-ordered. In addition, we found that surface RyR clusters are smaller both in extent and membership than those in the cell interior.Cav1.2 was commonly found in small spherical clusters of between 30-100 nm in diameter and were smaller and far denser than their RyR counterparts, with their centers appearing to be tightly packed. Cav1.2 clusters on the surface of the cell seemed little different from those in the interior, although the surface density was much higher. Super-clusters, made of 3 or more individual clusters were observed throughout the cell.NCX was more widely distributed and than either RyR or Cav1.2 and often formed a dense carpet on the cell surface made up of many small spherical clusters (∼50 nm dia). The mouth of the t-tubules were often very heavily labelled for 2-3 µm into the cell.

L-type calcium channel blockers and substance P induce angiogenesis of cortical vessels associated with beta-amyloid plaques in an Alzheimer mouse model

It is well established that L-type calcium channels (LTCCs) are expressed in astroglia. However, their functional role is still speculative, especially under pathologic conditions. We recently showed that the α1 subunit-like immunoreactivity of the CaV1.2 channel is strongly expressed in reactive astrocytes around beta-amyloid plaques in 11-month-old Alzheimer transgenic (tg) mice with the amyloid precursor protein London and Swedish mutations. The aim of the present study was to examine the cellular expression of all LTCC subunits around beta-amyloid plaques by in situ hybridization using 35S-labeled oligonucleotides. Our data show that messenger RNAs (mRNAs) of the LTCC CaV1.2 α1 subunit as well as all auxiliary β and α2δ subunits, except α2δ-4, were expressed in the hippocampus of age-matched wild-type mice. It was unexpected to see, that cells directly located in the plaque core in the cortex expressed mRNAs for CaV1.2 α1, β2, β4, and α2δ-1, whereas no expression was detected in the halo. Furthermore, cells in the plaque core also expressed preprotachykinin-A mRNA, the precursor for substance P. By means of confocal microscopy, we demonstrated that collagen-IV-stained brain vessels in the cortex were associated with the plaque core and were immunoreactive for substance P. In cortical organotypic brain slices of adult Alzheimer mice, we could demonstrate that LTCC blockers increased angiogenesis, which was further potentiated by substance P. In conclusion, our data show that brain vessels associated with beta-amyloid plaques express substance P and an LTCC and may play a role in angiogenesis.

L-type calcium channels in the auditory system

The voltage-activated L-type calcium channels Cav1.2 and Cav1.3 mediate Ca2+ influx into neurons at the soma or at dendrites, whereas they are not observed at the presynapse. Surprisingly, in the inner ear, Cav1.3 is indispensable for signal transmission from the presynaptic cochlear inner hair cells to the postsynaptic auditory nerve fibers. Due to Cav1.3 channel clustering at ribbons, i.e., specific presynaptic structures of the hair cells, they promote Ca2+ influx, which triggers calcium-dependent fusion of synaptic vesicles with the plasma membrane. Mutations in Cacna1d, a gene that encodes Cav1.3, result in deafness because release of the neurotransmitter glutamate at the synapses is abolished. Moreover, studies of the auditory pathway have revealed that Cav1.3 plays an important part in the central auditory system as well. Absence of the channel results in severe changes in auditory pathway cytoarchitecture and in abnormal electrophysiological performance of auditory neurons. Furthermore, developmental refinement of tonotopic inhibitory projections in sound localization circuits is disrupted. These aberrations are associated with abnormal sound processing in the auditory pathway. This goes to show that the Cav1.3 channel is essential for inner ear functioning as well as auditory pathway development and performance. Cacna1d therefore represents a prototypal deafness-associated gene, in which mutations result in both peripheral and central auditory deficiencies. This, in turn, has implications for auditory rehabilitation using cochlear implants that address only peripheral dysfunctions. Exploratory research into the closely related Cav1.2 isoform points to an important role of this channel in acoustic trauma. Cav1.2 is mainly expressed in the auditory nerve, but apparently not essential for normal auditory function. Loss of function of the channel, however, does influence the effects of traumatic noise exposure. Loss of this channel induced by noise trauma results in reduced auditory threshold increase—as compared with the control group. This phenomenon points to the fact that Cav1.2-mediated Ca2+ influx is involved in noise trauma-induced damage. Deeper insight into this function might result in new therapeutic approaches.