Increase In Cav1 Research Articles

CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer

Androgen deprivation therapy (ADT) is the main treatment for advanced prostate cancer (PCa) but resistance results in progression to terminal castrate resistant PCa (CRPC), where there is an unmet therapeutic need. Aberrant intracellular calcium (Cai2+) is known to promote neoplastic transformation and treatment resistance. There is growing evidence that voltage gated calcium channel (VGCC) expression is increased in cancer, particularly CACNA1D/CaV1.3 in CRPC. The aim of this study was to investigate if increased CaV1.3 drives resistance to ADT and determine its associated impact on Cai2+ and cancer biology. Bioinformatic analysis revealed that CACNA1D gene expression is increased in ADT treated PCa patients. This was corroborated in both in vivo LNCaP xenograft mouse and in vitro PCa cell line models, which demonstrated a significant increase in CaV1.3 protein expression following ADT with bicalutamide. Expression was found to be of a shortened 170kDa CaV1.3 isoform associated with plasma and intracellular membranes, which failed to induce calcium influx following membrane depolarisation. Instead, under ADT CaV1.3 mediated a rise in basal cytosolic calcium and an increase in store operated calcium entry (SOCE). This mechanism was found to promote the proliferation and survival of ADT resistant CRPC cells. Overall, this study demonstrates for the first time in PCa that under ADT specific CaV1.3 isoforms promote an upregulation of SOCE which contributes to treatment resistance and CRPC biology. Thus, this novel oncochannel represents a target for therapeutic development to improve PCa patient outcomes.

PDE1 Inhibition Modulates Cav1.2 Channel to Stimulate Cardiomyocyte Contraction.

[Figure: see text].

Chronic Intermittent Access to Alcohol Increases Ca v 1.2 in Dopamine Cells of the Substantia Nigra Pars Compacta

The high comorbidity of alcohol and methamphetamine (Meth) abuse presents unique toxicological endpoints. The serial exposure to chronic alcohol and then a binge model of Meth results in the loss of dopamine cells from the substantia nigra pars compacta (SNpc) with evidence for calcium-mediated excitotoxicity that is absent with either drug alone. The objective of this study is to localize and quantify the regional distribution of the increases in Cav1.2 in the substantia nigra after a chronic alcohol consumption paradigm in Sprague Dawley rats. We hypothesize that increases in Cav1.2 will be primarily in the tyrosine hydroxylase (TH)-positive cells of the SNpc. Male Sprague Dawley rats were subjected to a 2-bottle choice of water and chronic alcohol (EtOH) consumption model (i.e., 28-day intermittent/every other day access to 10% EtOH). At 24h after the last day of EtOH consumption, 4% PFA was perfused intracardially, brains cryostat sectioned, and TH and Cav1.2 immunoreactivities of the sections were identified. Fluorescence images of the SNpc and Ventral Tegmental Area (VTA) were captured at 20x magnification and cells that displayed TH and Cav1.2 co-localized immunoreactivity were hand-counted by an investigator blinded to the treatments. Cav1.2 was co-expressed with 43% of TH positive cells in the SNpc and 66% of TH positive cells in the VTA of control water drinking groups. There was a significant increase (39.3 ± 0.5%, p<0.05) in TH-positive cells with Cav1.2-positive immunoreactivity in the SNpc of EtOH-drinking rats when compared to water drinkers. No such effect was identified with TH-positive cells of the VTA. The total number of TH positive cells did not change in the alcohol drinking group. Western blot analysis of Cav1.2 protein in tissue that contained the SNpc of EtOH-drinking rats also showed increases in Cav1.2 . The localized increase in TH-positive cells of the SNpc, and not the VTA suggests a particular sensitivity of this population of cells to the calcium-mediated events that occurs following the serial exposure to EtOH and Meth.

Control of Arterial Ca V 1.2 Clustering and Coupled Gating by a Single Amino Acid

The L-type Ca2+ channel CaV1.2 plays key roles in cell excitability, muscle contraction and gene expression. These channels have been shown to gate in unison (e.g. coupled gating) in several cell types. CaV1.2 coupling amplifies Ca2+ influx, which can regulate cell function. In arterial myocytes, CaV1.2 coupling is increased upon elevations in extracellular glucose (HG) and in cells from diabetic patients and animal models of diabetes. Yet, the mechanisms for induction of this gating modality in CaV1.2 channels are poorly understood. Here, we tested the hypothesis that phosphorylation of CaV1.2 at S1928 is critical for induction of CaV1.2 clustering, which contributes to CaV1.2 coupling, amplification of Ca2+ influx and arterial myocyte contraction in response to HG and diabetes. Using a multiscale approach, we found that HG induced an increase in CaV1.2-mediated activity, open probability, and coupling frequency and strength in WT arterial myocytes, but not cells from a mouse in which S1928 was mutated to alanine to prevent the phosphorylation of this site (e.g. S1928A). Wild type-like CaV1.2 response to HG was observed in arterial myocytes from a mouse in which S1700 (another key CaV1.2 phosphorylation site) was prevented (e.g. S1700A), thus underscoring the importance S1928 phosphorylation on CaV1.2 function. Super-resolution nanoscopy and proximity ligation assay (PLA) revealed an increase in CaV1.2 clustering at the surface membrane of WT, but not S1928A arterial myocytes after acute HG incubation. Similar HG-induced CaV1.2 biophysical and structural alterations were recapitulated in arterial myocytes from WT diabetic mice (streptozotocin model) and human with diabetes, and these changes were completely absent in cells from diabetic S1928A mice. These results suggest a key role for S1928 phosphorylation in modulating CaV1.2 spatial distribution and gating mode upon HG and diabetes. We propose that our work may lay the foundation for novel therapeutic strategies with single amino acid accuracy to correct channel function and vascular reactivity during pathological conditions.

The Role of Alcohol in Hippocampal Calcium Channel (Cav1.2) expression

Background and Hypothesis: Voltage-gated L-type calcium channels (Cav1.2 and Cav1.3) in the hippocampus play important roles in glutamatergic neurotransmission underlying memory and learning. Their overexpression has been implicated in neuroexcitatory cell death and disease states including chronic alcoholism. While increases in Cav1.2 gene expression have been reported in the hippocampus after chronic ethanol exposure in rats, the regional distribution of Cav1.2 protein after voluntary ethanol (EtOH) drinking has not been reported. We hypothesize that the expression of Cav1.2 channels within the hippocampus is increased by EtOH drinking in a region-specific manner. Methods: Male Sprague Dawley rats were allowed 28 days of intermittent access to a 10% EtOH solution. At 24 hours after the last exposure to EtOH, brains were collected and processed for immunohistochemistry. Cav1.2 associated immunofluorescent signal from subregions of the hippocampus was quantified using ImageJ analysis software. Results: Immunohistochemical results indicate that Cav1.2 immunoreactivity in the hippocampal stratum granulosum layer within the Dentate Gyrus and the stratum pyramidale layer within CA1 and CA3 regions was increased in response to EtOH treatment. There was no significant change in Cav1.2 immunoreactivity for the CA2 region. Conclusion: This study suggests that calcium signaling in subregions of the hippocampus is differentially affected by EtOH consumption that may contribute to eventual calcium-mediated apoptosis. Impact and Implications: Understanding the process of EtOH-induced hippocampal calcium signaling presents opportunities for understanding the consequences of chronic alcohol exposure related to hippocampal function including memory and learning, and possible interventional therapies for alcohol damage.

Chronic Alcohol Drinking Increases L‐Type Voltage‐Gated Calcium Channel Cav1.2 in the Substantia Nigra

Studies report that 77 % of methamphetamine (Meth)‐dependent adults in the United States also have alcohol use disorder. This comorbidity is associated with negative cognitive and behavioral effects. In addition, the serial exposure of alcohol and then methamphetamine (Meth) to adult male Sprague Dawley rats is neurotoxic and marked by a loss of tyrosine hydroxylase‐positive dopamine (DA) cells in the substantia nigra pars compacta (SNpc) that is absent after either drug alone. Dopamine cell loss was associated with caspase‐3 mediated apoptosis after Meth exposure to alcohol drinking rats; however, the effect of alcohol that sensitizes DA cells to Meth‐induced apoptosis remains unknown. Adult male Sprague Dawley rats had access to either two bottles of water or a choice of 10% alcohol and water every other day over 28 days. Rats with access to alcohol every other day had significantly increased L‐type voltage gated calcium channel Cav1.2 protein in the substantia nigra (SN) (33 ± 1.4 %) at 1 day after the last day of alcohol drinking compared to water drinking only controls (p&lt;0.05). The increases in Cav1.2 were region‐specific since no such changes were detected in tissue from other dopaminergic regions including the dorsal striatum, ventral tegmental area, and nucleus accumbens. Furthermore, the cyclooxygenase‐2 selective and prostaglandin synthesis inhibitor, nimesulide (7.5mg/kg i.p., b.i.d.) administered on days when alcohol was not available, blocked the increases in Cav1.2 at 1 day after the alcohol drinking paradigm. Nimesulide administered only during the alcohol drinking paradigm also blocked the loss of DA cells in the SN pars compacta measured 1 week after exposure of Meth (10 mg/kg q 2 hrs X 4 injections). Although previous studies by others showed chronic alcohol exposure increases Cav1.2 in other brain regions, this is the first evidence of increases in the SN and that the increases occur in a prostaglandin synthesis‐dependent manner. The results are consistent with the excitotoxic effects produced calcium dysregulation and suggest that prostaglandins and disruption of calcium homeostasis produced by voluntary alcohol drinking sensitize DA cells in the SN to the neurodegenerative effects produced by the serial exposure to alcohol and Meth.Support or Funding InformationThis work is supported by the National Institutes of Health [DA042737].

Modulation of Ca2+-induced Ca2+ release by ubiquitin protein ligase E3 component n-recognin UBR3 and 6 in cardiac myocytes

ABSTRACT Ca2+-induced Ca2+ release (CICR) from sarcoplasmic reticulum is a finely tuned process responsible for cardiac excitation and contraction. The ubiquitin–proteasome system (UPS) as a major degradative system plays a crucial role in the maintenance of Ca2+ homeostasis. The E3 component N-recognin (UBR) subfamily is a part of the UPS; however, the role of UBR in regulating cardiac CICR is unknown. In the present study, we found that among the UBR family, single knockdown of UBR3 or UBR6 significantly elevated the amplitude of sarcoplasmic reticulum Ca2+ release without affecting Ca2+ transient decay time in neonatal rat ventricular myocytes. The protein expression of alpha 1 C subunit of L-type voltage-dependent Ca2+ channel (Cav1.2) was increased after UBR3/6 knockdown, whereas the protein levels of RyR2, SERCA2a, and PLB remained unchanged. In line with the increase in Cav1.2 proteins, the UBR3/6 knockdown enhanced the current of Cav1.2 channels. Furthermore, the increase in Cav1.2 proteins caused by UBR3/6 reduction was not counteracted by a protein biosynthesis inhibitor, cycloheximide, suggesting a degradative regulation of UBR3/6 on Cav1.2 channels. Our results indicate that UBR3/6 modulates cardiac CICR via targeting Cav1.2 protein degradation.

Increased caveolin-1 expression enhances the receptor-operated Ca2+ entry in the aorta of two-kidney, one-clip hypertensive rats.

What is the central question of this study? The aim was to examine and compare the contributions of caveolin-1 to the contractile responses mediated by L-type voltage-dependent calcium channels, store-operated Ca2+ channels and receptor-operated Ca2+ channels in two different types of arteries from two-kidney, one-clip hypertensive rats. What is the main finding and its importance? We demonstrated that the density of caveolae and caveolin-1 expression were significantly upregulated in the aorta of two-kidney, one-clip hypertensive rats, but not in the third-order branches of mesenteric arteries. We highlight that caveolin-1 plays an important role in aortic constriction by enhancing receptor-operated Ca2+ entry in the hypertensive rat model. Calcium and its multiple regulatory mechanisms are crucial for the development of hypertension. Among these regulatory mechanisms, store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE) mediate agonist-induced calcium influx, contributing to vascular contraction. The SOCE and ROCE are regulated by a variety of mechanisms involving caveolin-1 (Cav1), which has been found to be strongly associated with hypertension in gene polymorphism. In the present study, we investigated the role of Cav1 during the enhanced activity of calcium channels in hypertensive arteries. We demonstrated that the expression level of Cav1 was significantly increased in the aorta of two-kidney, one-clip (2K1C) hypertensive rats. The disruption of caveolae by methyl-β-cyclodextrin did not cause a marked difference in agonist-induced vasoconstriction in the third-order branches of the mesenteric arteries but strongly suppressed the aortic contractile response to endothelin-1 in the 2K1C group, which was not found in the control group. The increase in Cav1 by introduction of Cav1 scaffolding domain enhancing peptide promoted the 1-oleoyl-2-acetyl-glycerol-induced ROCE in hypertensive aortic smooth muscle cells but did not enhance the cyclopiazonic acid-induced SOCE. In the resistance arteries, similar changes were not observed, and no statistical changes of Cav1 expression were evident in the third-order branches of the mesenteric arteries. Our results indicate that increased Cav1 expression might promote the altered [Ca2+ ]i -induced aortic vasoreactivity by enhancing ROCE and be involved in the pathogenesis of hypertension.

Mechanical stretch increases L-type calcium channel stability in cardiomyocytes through a polycystin-1/AKT-dependent mechanism

The L-type calcium channel (LTCC) is an important determinant of cardiac contractility. Therefore, changes in LTCC activity or protein levels could be expected to affect cardiac function. Several studies describing LTCC regulation are available, but only a few examine LTCC protein stability. Polycystin-1 (PC1) is a mechanosensor that regulates heart contractility and is involved in mechanical stretch-induced cardiac hypertrophy. PC1 was originally described as an unconventional Gi/o protein-coupled receptor in renal cells. We recently reported that PC1 regulates LTCC stability in cardiomyocytes under stress; however, the mechanism underlying this effect remains unknown. Here, we use cultured neonatal rat ventricular myocytes and hypo-osmotic stress (HS) to model mechanical stretch. The model shows that the Cavβ2 subunit is necessary for LTCC stabilization in cardiomyocytes during mechanical stretch, acting through an AKT-dependent mechanism. Our data also shows that AKT activation depends on the G protein-coupled receptor activity of PC1, specifically its G protein-binding domain, and the associated Gβγ subunit of a heterotrimeric Gi/o protein. In fact, over-expression of the human PC1 C-terminal mutant lacking the G protein-binding domain blunted the AKT activation-induced increase in Cav1.2 protein in cardiomyocytes. These findings provide novel evidence that PC1 is involved in the regulation of cardiac LTCCs through a Giβγ-AKT-Cavβ2 pathway, suggesting a new mechanism for regulation of cardiac function.

Inhibition of L-Type Ca2+ Channels by TRPC1-STIM1 Complex Is Essential for the Protection of Dopaminergic Neurons.

Loss of dopaminergic (DA) neurons leads to Parkinson's disease; however, the mechanism(s) for the vulnerability of DA neurons is(are) not fully understood. We demonstrate that TRPC1 regulates the L-type Ca2+ channel that contributes to the rhythmic activity of adult DA neurons in the substantia nigra region. Store depletion that activates TRPC1, via STIM1, inhibits the frequency and amplitude of the rhythmic activity in DA neurons of wild-type, but not in TRPC1-/-, mice. Similarly, TRPC1-/- substantia nigra neurons showed increased L-type Ca2+ currents, decreased stimulation-dependent STIM1-Cav1.3 interaction, and decreased DA neurons. L-type Ca2+ currents and the open channel probability of Cav1.3 channels were also reduced upon TRPC1 activation, whereas increased Cav1.3 currents were observed upon STIM1 or TRPC1 silencing. Increased interaction between Cav1.3-TRPC1-STIM1 was observed upon store depletion and the loss of either TRPC1 or STIM1 led to DA cell death, which was prevented by inhibiting L-type Ca2+ channels. Neurotoxins that mimic Parkinson's disease increased Cav1.3 function, decreased TRPC1 expression, inhibited Tg-mediated STIM1-Cav1.3 interaction, and induced caspase activation. Importantly, restoration of TRPC1 expression not only inhibited Cav1.3 function but increased cell survival. Together, we provide evidence that TRPC1 suppresses Cav1.3 activity by providing an STIM1-based scaffold, which is essential for DA neuron survival.SIGNIFICANCE STATEMENT Ca2+ entry serves critical cellular functions in virtually every cell type, and appropriate regulation of Ca2+ in neurons is essential for proper function. In Parkinson's disease, DA neurons are specifically degenerated, but the mechanism is not known. Unlike other neurons, DA neurons depend on Cav1.3 channels for their rhythmic activity. Our studies show that, in normal conditions, the pacemaking activity in DA neurons is inhibited by the TRPC1-STIM1 complex. Neurotoxins that mimic Parkinson's disease target TRPC1 expression, which leads to an abnormal increase in Cav1.3 activity, thereby causing degeneration of DA neurons. These findings link TRPC1 to Cav1.3 regulation and provide important indications about how disrupting Ca2+ balance could have a direct implication in the treatment of Parkinson's patients.

Cav1.2 channels mediate persistent chronic stress-induced behavioral deficits that are associated with prefrontal cortex activation of the p25/Cdk5-glucocorticoid receptor pathway

Chronic stress is known to precipitate and exacerbate neuropsychiatric symptoms, and exposure to stress is particularly pathological in individuals with certain genetic predispositions. Recent genome wide association studies have identified single nucleotide polymorphisms (SNPs) in the gene CACNA1C, which codes for the Cav1.2 subunit of the L-type calcium channel (LTCC), as a common risk variant for multiple neuropsychiatric conditions. Cav1.2 channels mediate experience-dependent changes in gene expression and long-term synaptic plasticity through activation of downstream calcium signaling pathways. Previous studies have found an association between stress and altered Cav1.2 expression in the brain, however the contribution of Cav1.2 channels to chronic stress-induced behaviors, and the precise Cav1.2 signaling mechanisms activated are currently unknown. Here we report that chronic stress leads to a delayed increase in Cav1.2 expression selectively within the prefrontal cortex (PFC), but not in other stress-sensitive brain regions such as the hippocampus or amygdala. Further, we demonstrate that while Cav1.2 heterozygous (Cav1.2+/−) mice show chronic stress-induced depressive-like behavior, anxiety-like behavior, and deficits in working memory 1–2 days following stress, they are resilient to the effects of chronic stress when tested 5–7 days later. Lastly, molecular studies find a delayed upregulation of the p25/Cdk5-glucocorticoid receptor (GR) pathway in the PFC when examined 8 days post-stress that is absent in Cav1.2+/− mice. Our findings reveal a novel Cav1.2-mediated molecular mechanism associated with the persistent behavioral effects of chronic stress and provide new insight into potential Cav1.2 channel mechanisms that may contribute to CACNA1C-linked neuropsychiatric phenotypes.

The influence of lipoic acid on caveolin-1-regulated antioxidative enzymes in the mouse model of acute ulcerative colitis.

This study was undertaken to verify if two-weeks treatment of lipoic acid (LA) influence colon damage and pro-inflammatory cytokine synthesis during DSS-induced acute colitis. Moreover, as LA has anti-oxidative properties, we analyzed its influence on the level of antioxidative enzymes, HO-1 and eNOS, and their regulator- caveolin-1. LA was administrated to male C57/BALBc mice at a dose of 25 or 50mg/kg/day (i.p.) for 21days. Acute colitis was induced by administration of 4% DSS (w/v) in drinking water for 5days, followed by 2days of normal drinking water. Mice in LA+DSS groups were treated with LA (25 or 50mg/kg/day; i.p.) starting 14days prior to 4% DSS. Control group received saline for 21days. In the colon tissue we measured myeloperoxidase activity (MPO), IL-1β, IL-6, IL-17A, IL-23 (ELISA method), and tissue level of cav-1, phospho-eNOS, total eNOS and HO-1 (Western blot). Administration of DSS significantly increased total colon damage (p<0.001), myeloperoxidase (MPO) activity (p<0.05) and pro-inflammatory IL-6 (p<0.05). There was also a tendency towards higher IL-1β, IL-17A, and IL-23 in the colon. LA alone did not influence total colon damage, MPO activity, and pro-inflammatory cytokines concentration compared to control (p<0.05). Notably, mice treated with LA and DSS had significantly decreased total colon damage score (p<0.001), despite augmented colon MPO activity (p<0.01), but similar (IL-17A) or even significantly higher level (IL-1β, IL-23) as compared to the DSS group (p<0.05). IL-6 was insignificantly decreased after LA treatment at a dose of 50mg/kg. In acute colitis there was a tendency towards an increase in cav-1 and HO-1 and a decrease p-eNOS/total eNOS ratio. Moreover, the LA+DSS groups had higher expression of HO-1 and p-eNOS/total eNOS (p<0.05) compared to the DSS group, and a tendency towards higher cav-1 level. The changes did not depend on LA dose. Our study indicated that LA, at lower doses, may influence cav-1-regulated antioxidative enzyme levels (HO-1 and p-eNOS/total eNOS) despite an increase in colon pro-inflammatory cytokine levels during acute colitis. Hence, LA treatment may be - to some extent - beneficial in attenuation of acute colitis.

Spatial And Functional Coupling of The L‐Type Ca2+ Channel Cav1.2 with Ca2+‐Induced Ca2+ Release And cAMP Accumulation in INS‐1 cells

Exposure of pancreatic β‐cells to glucose generates concomitant oscillations in Ca2+ and cAMP which regulate insulin secretion, an essential function of β‐cells that promotes glucose homeostasis. Ca2+ influx through the L‐type Ca2+ channels Cav1.2 and Cav1.3 is further amplified by Ca2+ released from the ER via the ryanodine receptor (RyR) through a process called Ca2+‐induced Ca2+ release (CICR). L‐type Ca2+ channel blockers abolish glucose‐stimulated cAMP accumulation (GS‐cAMP); however, the role of CICR in this process is not well understood. We've demonstrated that expression of the Cav1.2 II‐III loop, a highly divergent region between the amino acid sequences of Cav1.2 and Cav1.3 that is required for critical protein‐protein interactions, displaces endogenous Cav1.2 channels from lipid raft domains in INS‐1 cells (Cav1.2/II‐III cells). Given our finding that Cav1.2 colocalizes with the ER Ca2+ release channel RyR2 in INS‐1 cells, expression of the Cav1.2 II‐III loop may perturb proper spatial coupling of Cav1.2 with RyR. Consistent with this, we found that CICR is uncoupled from Cav1.2‐mediated Ca2+ influx in Cav1.2/II‐III cells. To examine the role of CICR in GS‐cAMP, we expressed the Epac1‐based, cytosolic FRET sensor H187 in control INS‐1 and CICR‐deficient Cav1.2/II–III cells. When cAMP production was stimulated with the adenylyl cyclase activator forskolin, a robust response was detected in both control INS‐1 and Cav1.2/II–III cells, and there was no significant difference between them. In response to glucose (18 mM), we detected cAMP accumulation above baseline in control INS‐1 cells; however, glucose failed to elicit a detectable increase in Cav1.2/II–III cells. Further, both the L‐type Ca2+ channel blocker nicardipine (2 μM) and RyR inhibitor ryanodine (20 μM) completely abolished GS‐cAMP in control INS‐1 cells, suggesting that Ca2+ influx through L‐type Ca2+ channels and CICR are required for GS‐cAMP. Use of a plasma membrane‐targeted, Epac2‐based FRET sensor revealed that in Cav1.2/II–III cells, L‐type Ca2+ channel‐dependent GS‐cAMP is present at the plasma membrane. Taken together, Ca2+ influx through L‐type Ca2+ channels is sufficient to cause local GS‐cAMP; however, CICR largely amplifies the signal. Given our observation that GS‐cAMP is diminished in Cav1.2/II–III cells, we predicted that Cav1.2‐mediated CICR is preferentially coupled with GS‐cAMP. To test this, we utilized INS‐1 cells stably expressing dihydropyridine‐insensitive (DHPi) Cav1.2 and Cav1.3 channels. Here, the introduced DHPi channel is resistant to the DHP isradipine (2 μM); however, it remains sensitive to diltiazem (500 μM). As expected, we found that glucose markedly stimulated cAMP accumulation in both Cav1.2/DHPi and Cav1.3/DHPi cells. Surprisingly, isradipine significantly but incompletely inhibited GS‐cAMP in both cell lines, whereas diltiazem completely abolished the responses. Thus, Cav1.2 or Cav1.3 can sustain GS‐cAMP in the absence of the corresponding channel. Overall, we conclude that both Ca2+ influx through L‐type Ca2+ channels and CICR are required for efficient GS‐cAMP, and both Cav1.2 and Cav1.3 are involved in this process.Support or Funding InformationThis work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Disease (R01 DK064736) (to Gregory Hockerman) and a Purdue Research Foundation Grant (to Evan Pratt and Gregory Hockerman).

Myocardial KChIP2 Expression in Guinea Pig Resolves an Expanded Electrophysiologic Role.

Cardiac ion channels and their respective accessory subunits are critical in maintaining proper electrical activity of the heart. Studies have indicated that the K+ channel interacting protein 2 (KChIP2), originally identified as an auxiliary subunit for the channel Kv4, a component of the transient outward K+ channel (Ito), is a Ca2+ binding protein whose regulatory function does not appear restricted to Kv4 modulation. Indeed, the guinea pig myocardium does not express Kv4, yet we show that it still maintains expression of KChIP2, suggesting roles for KChIP2 beyond this canonical auxiliary interaction with Kv4 to modulate Ito. In this study, we capitalize on the guinea pig as a system for investigating how KChIP2 influences the cardiac action potential, independent of effects otherwise attributed to Ito, given the endogenous absence of the current in this species. By performing whole cell patch clamp recordings on isolated adult guinea pig myocytes, we observe that knock down of KChIP2 significantly prolongs the cardiac action potential. This prolongation was not attributed to compromised repolarizing currents, as IKr and IKs were unchanged, but was the result of enhanced L-type Ca2+ current due to an increase in Cav1.2 protein. In addition, cells with reduced KChIP2 also displayed lowered INa from reduced Nav1.5 protein. Historically, rodent models have been used to investigate the role of KChIP2, where dramatic changes to the primary repolarizing current Ito may mask more subtle effects of KChIP2. Evaluation in the guinea pig where Ito is absent, has unveiled additional functions for KChIP2 beyond its canonical regulation of Ito, which defines KChIP2 as a master regulator of cardiac repolarization and depolarization.

Looking for answers to L-type calcium channels in the ageing brain (Commentary on Zanos etal.).

Looking for answers to L-type calcium channels in the ageing brain (Commentary on Zanos etal.).

Ammonium increases Ca(2+) signalling and upregulates expression of Cav1.2 gene in astrocytes in primary cultures and in the in vivo brain.

The primary aim of this study was to identify the effects of hyperammonaemia on functional expression of Cav1.2 L-type Ca(2+) channels in astroglia. Primary cultures of mouse astrocytes were used to study effects of chronic treatment (1-5 days) with ammonium chloride, at 1, 3 and 5 mm on depolarization-induced increases in free cytosolic Ca(2+) concentration ([Ca(2+)]i , measured with Fura-2 based microfluorimetry) in control conditions and following treatment with the L-type Ca(2+) channel inhibitor, nifedipine, or with ryanodine receptor inhibitor, ryanodine. Expression of Cav1.2 mRNA was identified with RT-PCR, whereas protein content was determined by Western blotting. Sustained hyperammonaemia in vivo was induced by daily injections of urease (33 units kg body weight(-1), i.p.) for 3 days. Depolarization-induced [Ca(2+)]i transients sensitive to nifedipine (peak of the response) and to ryanodine (plateau phase) were significantly increased in astrocytes chronically exposed to ammonium. The ammonium-induced increase in Ca(2+) influx in astrocytes resulted from an upregulation of Cav1.2 channel's expression detected at mRNA and protein levels. Increase in Cav1.2 expression was prevented by ouabain antagonist canrenone. Similar upregulation of Cav1.2 gene expression was found in the brains of adult mice subjected to intraperitoneal injection of urease. In transgenic mice tagged with an astrocyte-specific or neurone-specific markers and treated with intraperitoneal injections of urease, the fluorescence-activated cell sorting of neurones and astrocytes demonstrated that Cav1.2 mRNA expression was upregulated in astrocytes, but not in neurones. Ammonium-induced deregulation of astroglial Ca(2+) signalling, is, in part, associated with upregulation of Cav1.2 L-type calcium channels.

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.

Soma size and Cav1.3 channel expression in vulnerable and resistant motoneuron populations of the SOD1G93A mouse model of ALS.

Although the loss of motoneurons is an undisputed feature of amyotrophic lateral sclerosis (ALS) in man and in its animal models (SOD1 mutant mice), how the disease affects the size and excitability of motoneurons prior to their degeneration is not well understood. This study was designed to test the hypothesis that motoneurons in mutant SOD1G93A mice exhibit an enlargement of soma size (i.e., cross‐sectional area) and an increase in Cav1.3 channel expression at postnatal day 30, well before the manifestation of physiological symptoms that typically occur at p90 (Chiu et al. 1995). We made measurements of spinal and hypoglossal motoneurons vulnerable to degeneration, as well as motoneurons in the oculomotor nucleus that are resistant to degeneration. Overall, we found that the somata of motoneurons in male SOD1G93A mutants were larger than those in wild‐type transgenic males. When females were included in the two groups, significance was lost. Expression levels of the Cav1.3 channels were not differentiated by genotype, sex, or any interaction of the two. These results raise the intriguing possibility of an interaction between male sex steroid hormones and the SOD1 mutation in the etiopathogenesis of ALS.

Surface L‐type Ca2+ channel expression levels are increased in aged hippocampus

Age-related increase in L-type Ca2+ channel (LTCC) expression in hippocampal pyramidal neurons has been hypothesized to underlie the increased Ca2+ influx and subsequent reduced intrinsic neuronal excitability of these neurons that lead to age-related cognitive deficits. Here, using specific antibodies against Cav1.2 and Cav1.3 subunits of LTCCs, we systematically re-examined the expression of these proteins in the hippocampus from young (3 to 4 month old) and aged (30 to 32 month old) F344xBN rats. Western blot analysis of the total expression levels revealed significant reductions in both Cav1.2 and Cav1.3 subunits from all three major hippocampal regions of aged rats. Despite the decreases in total expression levels, surface biotinylation experiments revealed significantly higher proportion of expression on the plasma membrane of Cav1.2 in the CA1 and CA3 regions and of Cav1.3 in the CA3 region from aged rats. Furthermore, the surface biotinylation results were supported by immunohistochemical analysis that revealed significant increases in Cav1.2 immunoreactivity in the CA1 and CA3 regions of aged hippocampal pyramidal neurons. In addition, we found a significant increase in the level of phosphorylated Cav1.2 on the plasma membrane in the dentate gyrus of aged rats. Taken together, our present findings strongly suggest that age-related cognitive deficits cannot be attributed to a global change in L-type channel expression nor to the level of phosphorylation of Cav1.2 on the plasma membrane of hippocampal neurons. Rather, increased expression and density of LTCCs on the plasma membrane may underlie the age-related increase in L-type Ca2+ channel activity in CA1 pyramidal neurons.

Why Has It Taken So Long to Learn What We Still Don’t Know?

Ca2+ is an essential ion and second messenger that plays a central role in determining membrane excitability and contractility in heart, while also participating in diverse and fundamental activities, such as gene transcription, cell metabolism and survival, vesicle secretion, learning, and memory. Voltage-gated Ca2+ channels (CaVx) are the primary entry point for extracellular Ca2+ to cross cell membranes and gain access to the intracellular space. In ventricular myocytes, CaV1.2 is the most important pathway for Ca2+ entry and provides a trigger for release of intracellular Ca2+ from the sarcoplasmic reticulum that activates myofilament crossbridge formation to grade the strength of each heart beat. Thus, improved understanding of Ca2+ homeostatic mechanisms, generally, and CaV1.2 regulation, specifically, are important goals for cardiovascular science. Article, see p 871 The increase in CaV1.2 current (ICa) following adrenergic receptor agonist stimulation is a fundamental aspect of fight or flight physiology, but the molecular mechanisms underlying this pathway are incompletely understood. Although Ca2+ was identified as a necessary ingredient for myocardial excitability by experiments by Sydney Ringer, over 100 years ago,1 knowledge of the signaling pathways that enhance cardiac contraction, in part, by increasing ICa has accrued more recently. Highlights in this discovery time line include the concept of receptors for extracellular chemical signals by Alquist,2 the identity of cAMP as a second messenger by Hardman et al,3 and enzymatic catalysis of amino acid phosphorylation by the cAMP kinase (protein kinase A [PKA]) to modify protein function by Kemp et al.4 Pioneering work by many investigators, including Mikami et al5 who first cloned the pore forming α-subunit of CaV1.2 and De Jongh et al6 who identified key biochemical features of several …