Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

Ca_v3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca_v3.2 and modulation by redox agents. His¹⁹¹ is found on the extracellular face of the Ca_v3.2 channel on the IS3-S4 linker and is not conserved in other Ca_v3 channels. Mutation of the corresponding residue in Ca_v3.1 to histidine, Gln¹⁷², significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca_v3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3–S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.     

Mechanism underlying the block of human Cav3.2 T-type Ca2+ channels by benidipine, a dihydropyridine Ca2+ channel blocker

AimsBenidipine, a dihydropyridine Ca²⁺ channel blocker, has been reported to block T-type Ca²⁺ channels; however, the mechanism underlying this effect was unclear. In this study, we characterized the mechanism responsible for this blocking activity. Furthermore, the blocking activity was compared between two enantiomers of benidipine, (S, S)- and (R, R)-benidipine.Main methodsHuman Ca_v3.2 (hCa_v3.2) T-type Ca²⁺ channels stably expressed in the human embryonic kidney cell line, HEK-293, were studied in whole-cell patch-clamp recordings and Ca²⁺ mobilization assay.Key findingsIn whole-cell patch-clamp recordings, benidipine blocked hCa_v3.2 T-type Ca²⁺ currents elicited by depolarization to a comparable extent as efonidipine. The block was dependent on stimulation frequency and holding potential, but not test potential. Benidipine significantly shifted the steady-state inactivation curve to the hyperpolarizing direction, but had no effect on the activation curve. Benidipine prolonged the recovery from inactivation of hCa_v3.2 T-type Ca²⁺ channels without any effect on the kinetics of activation, inactivation, or deactivation. In the Ca²⁺ mobilization assay, benidipine was more potent than efonidipine in blocking Ca²⁺ influx through hCa_v3.2 T-type Ca²⁺ channels. (S, S)-Benidipine was more potent than (R, R)-benidipine in blocking hCa_v3.2 T-type Ca²⁺ currents, but there was no difference in blocking the Ca²⁺ influx.SignificanceWe have characterized the blocking activity of benidipine against hCa_v3.2 Ca²⁺ channels and revealed the difference between the two enantiomers of benidipine. The blocking action of benidipine could be mediated by stabilizing hCa_v3.2 Ca²⁺ channels in an inactivated state.     

T-type calcium channel expression and function in the diseased heart

The regulation of intracellular Ca²⁺ is essential for cardiomyocyte function, and alterations in proteins that regulate Ca²⁺ influx have dire consequences in the diseased heart. Low voltage-activated, T-type Ca²⁺ channels are one pathway of Ca²⁺ entry that is regulated according to developmental stage and in pathological conditions in the adult heart. Cardiac T-type channels consist of two main types, Ca_v3.1 (α1G) and Ca_v3.2 (α1H), and both can be induced in the myocardium in disease and injury but still, relatively little is known about mechanisms for their regulation and their respective functions. This article integrates previous data establishing regulation of T-type Ca²⁺ channels in animal models of cardiac disease, with recent data that begin to address the functional consequences of cardiac Ca_v3.1 and Ca_v3.2 Ca²⁺ channel expression in the pathological setting. The putative association of T-type Ca²⁺ channels with Ca²⁺ dependent signaling pathways in the context of cardiac hypertrophy is also discussed.     

Gαi2- and Gαi3-specific regulation of voltage-dependent L-type calcium channels in cardiomyocytes

Background

Two pertussis toxin sensitive G_i proteins, G_i2 and G_i3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G_i isoforms are functionally distinct. To test for isoform-specific functions of G_i proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC).

Methods

Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gα_i2 (Gα_i2 ^−/−) or Gα_i3 (Gα_i3 ^−/−). mRNA levels of Gα_i/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gα_i and Ca_vα₁ protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings.

Results

In cardiac tissue from Gα_i2 ^−/− mice, Gα_i3 mRNA and protein expression was upregulated to 187±21% and 567±59%, respectively. In Gα_i3 ^−/− mouse hearts, Gα_i2 mRNA (127±5%) and protein (131±10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gα_i2 ^−/− mice was lowered (−7.9±0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (−10.7±0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gα_i3 ^−/− mice (−14.3±0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gα_i2 (but not of Gα_i3) and following treatment with pertussis toxin in Gα_i3 ^−/−. The pore forming Ca_vα₁ protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca_vα₁ and Ca_vβ₂ subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gα_i2.

Conclusion

Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gα_i proteins. In particular, loss of Gα_i2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.     

Osteopenia due to enhanced cathepsin K release by BK channel ablation in osteoclasts

Background

The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified.

Methodology/Principal Findings

We found, that in juvenile bone the large conductance, voltage and Ca²⁺-activated (BK) K⁺ channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K⁺ outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK^−/−) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK^−/− vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca²⁺ and triiodthyronine as well as osteoclastogenesis were not altered in BK^−/− females.

Conclusion/Significance

Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK^−/− mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity.     

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells

Inactivation of inward-rectifying K⁺ channels (I_K,in) by a rise in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca²⁺]_i action on I_K,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca²⁺]_i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording I_K,in under a voltage clamp and [Ca²⁺]_i by Fura-2 fluorescence ratiophotometry. I_K,in inactivation correlated positively with [Ca²⁺]_i and indicated a K_i of 329 ± 31 nm with cooperative binding of four Ca²⁺ ions per channel. I_K,in was promoted by the Ca²⁺ channel antagonists Gd³⁺ and calcicludine, both of which suppressed the [Ca²⁺]_i rise, but the [Ca²⁺]_i rise was unaffected by the K⁺ channel blocker Cs⁺. We also found that ryanodine, an antagonist of intracellular Ca²⁺ channels that mediate Ca²⁺-induced Ca²⁺ release, blocked the [Ca²⁺]_i rise, and Mn²⁺ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca²⁺]_i control of I_K,in and to roles for discrete Ca²⁺ flux pathways in feedback control of the K⁺ channels by membrane voltage.Ca²⁺ underlies many fundamental regulatory processes in plants, including adaptive responses to abiotic environmental stress (Knight et al., 1996; Russell et al., 1996; McAinsh et al., 1997) and programmed cell death evoked by pathogen attack (Low and Merida, 1996; Hammondkosack and Jones, 1997). Coordination of changes in [Ca²⁺]_i and its integration with downstream response elements are central in coupling stimulus input to cellular response in these processes.In stomatal guard cells, the best characterized higher-plant cell model, major downstream targets of [Ca²⁺]_i and their roles in stomatal function have been identified. Increasing [Ca²⁺]_i is known to inactivate I_K,in and to activate Cl⁻ channels, events that bias plasma membrane transport for net efflux of osmotically active solute and a loss of turgor, which drives stomatal closure (Blatt and Grabov, 1997). Furthermore, changes in [Ca²⁺]_i are associated with ABA, CO₂, and the growth hormone auxin (Blatt and Grabov, 1997; McAinsh et al., 1997). These [Ca²⁺]_i signals have been observed to oscillate (McAinsh et al., 1995; Webb et al., 1996), characteristics that may constitute “Ca²⁺ signatures” to encode specific downstream responses (Berridge, 1996). Yet, despite the evidence for [Ca²⁺]_i signaling in guard cells, surprisingly little detail is known about the link between [Ca²⁺]_i changes and ion channel activity at the plasma membrane or about the mechanisms mediating such [Ca²⁺]_i changes. To our knowledge, in no instance have the characteristics of ion channel regulation by Ca²⁺ been quantified directly in any higher-plant cell.We recently described the coupling of membrane voltage to [Ca²⁺]_i, demonstrating that hyperpolarization, whether under a voltage clamp or in the presence of low [K⁺]_o, evoked [Ca²⁺]_i increases in guard cells, and that the voltage threshold for [Ca²⁺]_i rise was profoundly altered by ABA (Grabov and Blatt, 1998). Our observations indicated a link to Ca²⁺ influx across the plasma membrane and raised questions about the efficacy of [Ca²⁺]_i in inactivating I_K,in and about the contributions of intracellular Ca²⁺ release to the [Ca²⁺]_i signal. We have used membrane voltage to experimentally manipulate [Ca²⁺]_i and report that I_K,in is strongly dependent on [Ca²⁺]_i, consistent with a cooperative binding of four Ca²⁺ ions to effect inactivation. Additional experiments indicate that voltage-evoked [Ca²⁺]_i increases depend both on Ca²⁺ influx and on release of Ca²⁺ from intracellular stores. These results underscore the role of [Ca²⁺]_i as a high-gain “switch” in the control of I_K,in, and implicate [Ca²⁺]_i in feedback control linking membrane voltage to the activity of the K⁺ channels.     

Selectivity signatures of three isoforms of recombinant T-type Ca channels

Voltage-gated Ca²⁺ channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca²⁺ over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca²⁺ permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca²⁺-channel α1 subunits, Ca_v3.1, Ca_v3.2 and Ca_v3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca_v3.1, Ca_v3.2, and Ca_v3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca_v3 channels select Ca²⁺ over Na⁺ by affinity. Ca_v3.1 and Ca_v3.2 discriminate Ca²⁺, Sr²⁺ and Ba²⁺ based on the ion's effects on the open channel probability, whilst Ca_v3.3 discriminates based on the ion's intrapore binding affinity. All Ca_v3s were characterized by much smaller difference in the K_D values for Na⁺ current blockade by Ca²⁺ (K_D1 ∼ 6 μM) and for Ca²⁺ current saturation (K_D2 ∼ 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na⁺/Ca²⁺ current at close to physiological Ca²⁺ concentrations, which was the strongest for Ca_v3.3, smaller for Ca_v3.2 and the smallest for Ca_v3.1. In addition to intrapore Ca²⁺ binding site(s) Ca_v3.2, but not Ca_v3.1 and Ca_v3.3, is likely to possess an extracellular Ca²⁺ binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca²⁺ current in native cells.     

Synthesis and biological evaluation of pyrrolidine-based T-type calcium channel inhibitors for the treatment of neuropathic pain

The treatment of neuropathic pain is one of the urgent unmet medical needs and T-type calcium channels are promising therapeutic targets for neuropathic pain. Several potent T-type channel inhibitors showed promising in vivo efficacy in neuropathic pain animal models and are being investigated in clinical trials. Herein we report development of novel pyrrolidine-based T-type calcium channel inhibitors by pharmacophore mapping and structural hybridisation followed by evaluation of their Ca_v3.1 and Ca_v3.2 channel inhibitory activities. Among potent inhibitors against both Ca_v3.1 and Ca_v3.2 channels, a promising compound 20n based on in vitro ADME properties displayed satisfactory plasma and brain exposure in rats according to in vivo pharmacokinetic studies. We further demonstrated that 20n effectively improved the symptoms of neuropathic pain in both SNL and STZ neuropathic pain animal models, suggesting modulation of T-type calcium channels can be a promising therapeutic strategy for the treatment of neuropathic pain.     

Akt regulates L-type Ca2+ channel activity by modulating Cavα1 protein stability

The insulin IGF-1–PI3K–Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca²⁺ handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca²⁺ channel (LTCC) protein density. The pore-forming channel subunit Ca_vα1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Ca_vα1 protein levels. Our findings show that Akt-dependent phosphorylation of Ca_vβ2, the LTCC chaperone for Ca_vα1, antagonizes Ca_vα1 protein degradation by preventing Ca_vα1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca²⁺ channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca²⁺ entry, Ca²⁺ handling, and contractility.     

The plasma membrane channel ORAI1 mediates detrimental calcium influx caused by endogenous oxidative stress

The mouse hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Addition of extracellular glutamate depletes the cells of glutathione (GSH) by blocking the glutamate−cystine antiporter system x_c⁻. GSH is the main antioxidant in neurons and its depletion induces a well-defined program of cell death called oxytosis, which is probably synonymous with the iron-dependent form of non-apoptotic cell death termed ferroptosis. Oxytosis is characterized by an increase of reactive oxygen species and a strong calcium influx preceding cell death. We found a significant reduction in store-operated calcium entry (SOCE) in glutamate-resistant HT22 cells caused by downregulation of the Ca²⁺ channel ORAI1, but not the Ca²⁺ sensors STIM1 or STIM2. Pharmacological inhibition of SOCE mimicked this protection similarly to knockdown of ORAI1 by small interfering RNAs. Long-term calcium live-cell imaging after induction of the cell death program showed a specific reduction in Ca²⁺-positive cells by ORAI1 knockdown. These results suggest that dysregulated Ca²⁺ entry through ORAI1 mediates the detrimental Ca²⁺ entry in programmed cell death induced by GSH depletion. As this detrimental Ca²⁺ influx occurs late in the course of the cell death program, it might be amenable to therapeutic intervention in diseases caused by oxidative stress.     

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a +/? Mouse Model

Background

Loss-of-function mutations in SCN5A, the gene encoding Na_v1.5 Na⁺ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a ^+/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/Principal Findings

Based on ECG, 10-week-old Scn5a ^+/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na⁺ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a ^+/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a ^+/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a ^+/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a ^+/− mice had similar Na_v1.5 mRNA but higher Na_v1.5 protein expression, and moderately larger I_Na current than severely affected Scn5a ^+/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a ^+/− mice than in mildly affected ones.

Conclusions

Scn5a ^+/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a ^+/− mice, phenotype severity correlates with wild-type Na_v1.5 protein expression.     

Enhancement effects of martentoxin on glioma BK channel and BK channel (α+β1) subtypes

Background

BK channels are usually activated by membrane depolarization and cytoplasmic Ca²⁺. Especially,the activity of BK channel (α+β4) can be modulated by martentoxin, a 37 residues peptide, with Ca²⁺-dependent manner. gBK channel (glioma BK channel) and BK channel (α+β1) possessed higher Ca²⁺ sensitivity than other known BK channel subtypes.

Methodology and Principal Findings

The present study investigated the modulatory characteristics of martentoxin on these two BK channel subtypes by electrophysiological recordings, cell proliferation and Ca²⁺ imaging. In the presence of cytoplasmic Ca²⁺, martentoxin could enhance the activities of both gBK and BK channel (α+β1) subtypes in dose-dependent manner with EC₅₀ of 46.7 nM and 495 nM respectively, while not shift the steady-state activation of these channels. The enhancement ratio of martentoxin on gBK and BK channel (α+β1) was unrelated to the quantitive change of cytoplasmic Ca²⁺ concentrations though the interaction between martentoxin and BK channel (α+β1) was accelerated under higher cytoplasmic Ca²⁺. The selective BK pore blocker iberiotoxin could fully abolish the enhancement of these two BK subtypes induced by martentoxin, suggesting that the auxiliary β subunit might contribute to the docking for martentoxin. However, in the absence of cytoplasmic Ca²⁺, the activity of gBK channel would be surprisingly inhibited by martentoxin while BK channel (α+β1) couldn''t be affected by the toxin.

Conclusions and Significance

Thus, the results shown here provide the novel evidence that martentoxin could increase the two Ca²⁺-hypersensitive BK channel subtypes activities in a new manner and indicate that β subunit of these BK channels plays a vital role in this enhancement by martentoxin.     

Pituitary adenylate cyclase-activating peptide (PACAP) recruits low voltage-activated T-type calcium influx under acute sympathetic stimulation in mouse adrenal chromaffin cells

Low voltage-activated T-type Ca_v3.2 calcium channels are expressed in neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that naïve adrenal chromaffin cells express a nominal Ca_v3.2-dependent conductance. However, Ca_v3.2 conductance is up-regulated following chronic hypoxia or long term exposure to cAMP analogs. Thus, although a link between chronic stressors and up-regulation of Ca_v3.2 exists, there are no reports testing the specific role of Ca_v3.2 channels in the acute sympathoadrenal stress response. In this study, we examined the effects of acute sympathetic stress on T-type Ca_v3.2 calcium influx in mouse chromaffin cells in situ. Pituitary adenylate cyclase-activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. PACAP stimulation did not evoke action potential firing in chromaffin cells but did cause a persistent subthreshold membrane depolarization that resulted in an immediate and robust Ca²⁺-dependent catecholamine secretion. Moreover, PACAP-evoked secretion was sensitive to block by nickel chloride and was acutely inhibited by protein kinase C blockers. We utilized perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism of PACAP-evoked calcium entry. We provide evidence that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phosphodependent recruitment of a T-type Ca_v3.2 Ca²⁺ influx. This in turn evokes catecholamine release during the acute sympathetic stress response.     

Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca²⁺ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca²⁺ selective filters of all high-voltage-activated Ca²⁺ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca²⁺ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca_v3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca_v3.2, including relative permeability between Ba²⁺ and Ca²⁺, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd²⁺ blocking sensitivity, and pH effects, in distinctive ways.     

Altered synaptic plasticity and behavioral abnormalities in CNGA3‐deficient mice

The role of the cyclic nucleotide‐gated (CNG) channel CNGA3 is well established in cone photoreceptors and guanylyl cyclase‐D‐expressing olfactory neurons. To assess a potential function of CNGA3 in the mouse amygdala and hippocampus, we examined synaptic plasticity and performed a comparative analysis of spatial learning, fear conditioning and step‐down avoidance in wild‐type mice and CNGA3 null mutants (CNGA3^?/?). CNGA3^?/? mice showed normal basal synaptic transmission in the amygdala and the hippocampus. However, cornu Ammonis (CA1) hippocampal long‐term potentiation (LTP) induced by a strong tetanus was significantly enhanced in CNGA3^?/? mice as compared with their wild‐type littermates. Unlike in the hippocampus, LTP was not significantly altered in the amygdala of CNGA3^?/? mice. Enhanced hippocampal LTP did not coincide with changes in hippocampus‐dependent learning, as both wild‐type and mutant mice showed a similar performance in water maze tasks and contextual fear conditioning, except for a trend toward higher step‐down latencies in a passive avoidance task. In contrast, CNGA3^?/? mice showed markedly reduced freezing to the conditioned tone in the amygdala‐dependent cued fear conditioning task. In conclusion, our study adds a new entry on the list of physiological functions of the CNGA3 channel. Despite the dissociation between physiological and behavioral parameters, our data describe a so far unrecognized role of CNGA3 in modulation of hippocampal plasticity and amydgala‐dependent fear memory.     

Toll-like receptor-2 mediates diet and/or pathogen associated atherosclerosis: proteomic findings

Background

Accumulating evidence implicates a fundamental link between the immune system and atherosclerosis. Toll-like receptors are principal sensors of the innate immune system. Here we report an assessment of the role of the TLR2 pathway in atherosclerosis associated with a high-fat diet and/or bacteria in ApoE^+/− mice.

Methods and Results

To explore the role of TLR2 in inflammation- and infection-associated atherosclerosis, 10 week-old ApoE^+/−-TLR2^+/+, ApoE^+/−-TLR2^+/− and ApoE^+/−-TLR2^−/− mice were fed either a high fat diet or a regular chow diet. All mice were inoculated intravenously, once per week for 24 consecutive weeks, with 50 µl live Porphyromonas gingivalis (P.g) (10⁷ CFU) or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. ApoE^+/−-TLR2^+/+ mice showed a significant increase in atheromatous lesions in proximal aorta and aortic tree compared to ApoE^+/−-TLR2^+/− and ApoE^+/−-TLR2^−/− mice for all diet conditions. They also displayed profound changes in plaque composition, as evidenced by increased macrophage infiltration and apoptosis, increased lipid content, and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. SAA levels from ApoE^+/−-TLR2^+/+ mice were significantly higher than from ApoE^+/−-TLR2^+/− and ApoE^+/−-TLR2^−/− mice. Serum cytokine analysis revealed increased levels of pro-inflammatory cytokines in ApoE^+/−-TLR2^+/+ mice compared to ApoE^+/−-TLR2^+/− and TLR2^−/− mice, irrespective of diet or bacterial challenge. ApoE^+/−-TLR2^+/+ mice injected weekly for 24 weeks with FSL-1 (a TLR2 agonist) also demonstrated significant increases in atherosclerotic lesions, SAA and serum cytokine levels compared to ApoE^+/−-TLR2^−/− mice under same treatment condition. Finally, mass-spectrometry (MALDI-TOF-MS) of aortic samples analyzed by 2-dimentional gel electrophoresis differential display, identified 6 proteins upregulated greater than 2-fold in ApoE^+/−-TLR2^+/+ mice fed the high fat diet and inoculated with P.g compared to any other group.

Conclusion

Genetic deficiency of TLR2 reduces diet- and/or pathogen-associated atherosclerosis in ApoE^+/− mice, along with differences in plaque composition suggesting greater structural stability while TLR-2 ligand-specific activation triggers atherosclerosis. The present data offers new insights into the pathophysiological pathways involved in atherosclerosis and paves the way for new pharmacological interventions aimed at reducing atherosclerosis.