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In the microcirculation, longitudinal conduction of vasomotor responses provides an essential means of coordinating flow distribution among vessels in a complex network. Spread of current along the vessel axis can display a regenerative component, which leads to propagation of vasomotor signals over many millimeters; the ionic basis for the regenerative response is unknown. We examined the responses to 10 s of focal electrical stimulation (30 Hz, 2 ms, 30 V) of mouse cremaster arterioles to test the hypothesis that voltage-dependent Na(+) (Na(v)) and Ca(2+) channels might be activated in long-distance signaling in microvessels. Electrical stimulation evoked a vasoconstriction at the site of stimulation and a spreading, nondecremental conducted dilation. Endothelial damage (air bubble) blocked conduction of the vasodilation, indicating an involvement of the endothelium. The Na(v) channel blocker bupivacaine also blocked conduction, and TTX attenuated it. The Na(v) channel activator veratridine induced an endothelium-dependent dilation. The Na(v) channel isoforms Na(v)1.2, Na(v)1.6, and Na(v)1.9 were detected in the endothelial cells of cremaster arterioles by immunocytochemistry. These findings are consistent with the involvement of Na(v) channels in the conducted response. BAPTA buffering of endothelial cell Ca(2+) delayed and reduced the conducted dilation, which was almost eliminated by Ni(2+), amiloride, or deletion of alpha(1H) T-type Ca(2+) (Ca(v)3.2) channels. Blockade of endothelial nitric oxide synthase or Ca(2+)-activated K(+) channels also inhibited the conducted vasodilation. Our findings indicate that an electrically induced signal can propagate along the vessel axis via the endothelium and can induce sequential activation of Na(v) and Ca(v)3.2 channels. The resultant Ca(2+) influx activates endothelial nitric oxide synthase and Ca(2+)-activated K(+) channels, triggering vasodilation.

Ca(2+) channels play critical roles in the regulation of synaptic activity. In contrast to the well established function of voltage-activated Ca(2+) channels in the presynaptic membrane for neurotransmitter release, some studies are just beginning to elucidate the functions of the Ca(2+) channels in the postsynaptic membrane. In this study, we demonstrated the functional association of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors with the neuronal Ca(2+) channels. A series of biochemical studies showed the specific association of Ca(v)2.1 (alpha(1A)-class) and Ca(v)2.2 (alpha(1B)-class) with AMPA receptors in the postsynaptic membrane. Our electrophysiological and Ca(2+) imaging analyses of recombinant Ca(v)2.1 and AMPA receptors also showed functional coupling of the two channels. Considering the critical roles of postsynaptic intracellular concentration of Ca(2+) ([Ca(2+)](i)) increase and AMPA receptor trafficking for long-term potentiation (LTP) and long-term depression (LTD), the functional association of Ca(2+) channels with the AMPA receptors may provide new insights into the mechanism of synaptic plasticity.

The influence of the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis on expression of low-voltage-activated (LVA) Ca2+ current in atrial tissue was investigated using spontaneous dwarf (SpDwf) rats, a mutant strain that lacks GH. Atrial myocytes from SpDwf rats express LVA and high-voltage-activated (HVA) Ca2+ currents and the Ca2+ channel alpha1-subunit genes CaV1.2, CaV2.3, CaV3.1, and CaV3.2. LVA current density decreases significantly beginning at, or shortly after, birth in normal animals; however, its density is maintained in SpDwf rats at 1 pA/pF for > or =12 wk after birth. The abundance of mRNAs encoding CaV2.3 and CaV3.2 declines with advancing age in normal atrial development, yet expression of CaV2.3 mRNA remains significantly elevated in older SpDwf animals. Quantitation of local transcript levels for mRNAs encoding IGF-I and IGF-I receptor (IGF-IR) also reveals significant differences in expression of these transcripts in atrial tissue of SpDwf animals compared with controls. In SpDwf rats, the abundance of IGF-IR mRNA remains elevated at many postnatal ages, whereas mRNA encoding IGF-I is maintained only in older animals. Physiological concentrations of IGF-I cause two- to threefold increases in LVA current density in primary cultures of atrial myocytes, and this effect is blocked by an antisense oligonucleotide targeting the IGF-IR. Thus disruption of GH production in SpDwf animals alters expression of atrial LVA Ca2+ channel and IGF genes as well as postnatal regulation of LVA Ca2+ current density, most likely acting through compensatory mechanisms via the local IGF-IR.

Voltage-dependent calcium channels (VDCC) are multiprotein assemblies that regulate the entry of extracellular calcium into electrically excitable cells and serve as signal transduction centers. The alpha1 subunit forms the membrane pore while the intracellular beta subunit is responsible for trafficking of the channel to the plasma membrane and modulation of its electrophysiological properties. Crystallographic analyses of a beta subunit functional core alone and in complex with a alpha1 interaction domain (AID) peptide, the primary binding site of beta to the alpha1 subunit, reveal that beta represents a novel member of the MAGUK protein family. The findings illustrate how the guanylate kinase fold has been fashioned into a protein-protein interaction module by alteration of one of its substrate sites. Combined results indicate that the AID peptide undergoes a helical transition in binding to beta. We outline the mechanistic implications for understanding the beta subunit's broad regulatory role of the VDCC, particularly via the AID.

We characterized the neuronal two-domain (95kD-alpha(1)2.1) form of the alpha(1)2.1 subunit of the voltage-gated calcium channels using genetic and molecular analysis. The 95kD-alpha(1)2.1 is absent in neuronal preparations from CACNA1A null mouse demonstrating that alpha(1)2.1 and 95kD-alpha(1)2.1 arise from the same gene. A recombinant two-domain form (alpha(1AI-II)) of alpha(1)2.1 associates with the beta subunit and is trafficked to the plasma membrane. Translocation of the alpha(1AI-II) to the plasma membrane requires association with the beta subunit, since a mutation in the alpha(1AI-II) that inhibits beta subunit association reduces membrane trafficking. Though the alpha(1AI-II) protein does not conduct any voltage-gated currents, we have previously shown that it generates a high density of non-linear charge movements [Ahern et al., Proc. Natl. Acad. Sci. USA 98 (2001) 6935-6940]. In this study, we demonstrate that co-expression of the alpha(1AI-II) significantly reduces the current amplitude of alpha(1)2.1/beta(1a)/alpha(2)delta channels, via competition for the beta subunit. Taken together, our results demonstrate a dual functional role for the alpha(1AI-II) protein, both as a voltage sensor and modulator of P/Q-type currents in recombinant systems. These studies suggest an in vivo role for the 95kD-alpha(1)2.1 in altering synaptic activity via protein-protein interactions and/or regulation of P/Q-type currents.

The Cav3.2 T-type calcium channel is implicated in various pathological conditions, including cardiac hypertrophy, epilepsy, autism, and chronic pain. Phosphorylation of Cav3.2 by multiple kinases plays a pivotal role in regulating its calcium channel function. The calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin, interacts physically with Cav3.2 and modulates its activity. However, it remains unclear whether calcineurin dephosphorylates Cav3.2, the specific spatial regions on Cav3.2 involved, and the extent of the quantitative impact. In this study, we elucidated the serine/threonine residues on Cav3.2 targeted by calcineurin using quantitative mass spectrometry. We identified six serine residues in the N-terminus, II–III loop, and C-terminus of Cav3.2 that were dephosphorylated by calcineurin. Notably, a higher level of dephosphorylation was observed in the Cav3.2 C-terminus, where calcineurin binds to this channel. Additionally, a previously known CaMKII-phosphorylated site, S1198, was found to be dephosphorylated by calcineurin. Furthermore, we also discovered that a novel CaMKII-phosphorylated site, S2137, underwent dephosphorylation by calcineurin. In CAD cells, a mouse central nervous system cell line, membrane depolarization led to an increase in the phosphorylation of endogenous Cav3.2 at S2137. Mutation of S2137 affected the calcium channel function of Cav3.2. Our findings advance the understanding of Cav3.2 regulation not only through kinase phosphorylation but also via calcineurin phosphatase dephosphorylation. [ABSTRACT FROM AUTHOR]

Inhibition of T-type calcium channels (CaV3) prevents development of diseases related to cardiovascular and nerve systems. Further, knockout animal studies have revealed that some diseases are mediated by specific subtypes of CaV3. However, subtype-specific CaV3 inhibitors for therapeutic purposes or for studying the physiological roles of CaV3 subtypes are missing. To bridge this gap, we employed our spider venom library and uncovered that Avicularia spec. ("Amazonas Purple", Peru) tarantula venom inhibited specific T-type CaV channel subtypes. By using chromatographic and mass-spectrometric techniques, we isolated and sequenced the active toxin ω-Avsp1a, a C-terminally amidated 36 residue peptide with a molecular weight of 4224.91 Da, which comprised the major peak in the venom. Both native (4.1 μM) and synthetic ω-Avsp1a (10 μM) inhibited 90% of CaV3.1 and CaV3.3, but only 25% of CaV3.2 currents. In order to investigate the toxin binding site, we generated a range of chimeric channels from the less sensitive CaV3.2 and more sensitive CaV3.3. Our results suggest that domain-1 of CaV3.3 is important for the inhibitory effect of ω-Avsp1a on T-type calcium channels. Further studies revealed that a leucine of T-type calcium channels is crucial for the inhibitory effect of ω-Avsp1a. [ABSTRACT FROM AUTHOR]

Homozygous Cav3.2 knockout mice, which are defective in the pore-forming subunit of a low voltage activated T-type calcium channel, have been documented to show impaired maintenance of late-phase long-term potentiation (L-LTP) and defective retrieval of context-associated fear memory. To investigate the role of Cav3.2 in global gene expression, we performed a microarray transcriptome study on the hippocampi of the Cav3.2-/- mice and their wild-type littermates, either naïve (untrained) or trace fear conditioned. We found a significant left-right asymmetric effect on the hippocampal transcriptome caused by the Cav3.2 knockout. Between the naive Cav3.2-/- and the naive wild-type mice, 3522 differentially expressed genes (DEGs) were found in the left hippocampus, but only 4 DEGs were found in the right hippocampus. Remarkably, the effect of Cav3.2 knockout was partially reversed by trace fear conditioning. The number of DEGs in the left hippocampus was reduced to 6 in the Cav3.2 knockout mice after trace fear conditioning, compared with the wild-type naïve mice. To our knowledge, these results demonstrate for the first time the asymmetric effects of the Cav3.2 and its partial reversal by behavior training on the hippocampal transcriptome. [ABSTRACT FROM AUTHOR]

Aims The Cav3.2 T-channel plays a pivotal role in inducing calcineurin/nuclear factor of activated T cell (NFAT) signalling during cardiac hypertrophy. Because calcineurin/NFAT signalling is induced early after pressure overload, we hypothesized that Cav3.2 is induced by an early signal. Our aim is to investigate when and how Cav3.2 is induced during cardiac hypertrophy. Methods and results The evolutionary conserved promoter Cav3.2-3500 from mouse genome was validated to express the reporter gene as endogenous Cav3.2 in cell lines and transgenic (Tg; Cav3.2-3500-Luc) mice. The early induction of luciferase in Tg mice and Cav3.2 mRNA in wild-type mice after transverse aortic banding (TAB) surgery supported our hypothesis that Cav3.2 is induced early during cardiac hypertrophy. The TAB-responding element [−81 to −41 bp upstream of the transcription start site (TSS) of mouse Cav3.2] was identified by in vivo gene transfer by injecting reporter constructs into the left ventricle followed by TAB surgery. Electrophoresis mobility shift assay and chromatin immunoprecipitation assays revealed that Egr1 bound to the TAB-responding element of Cav3.2. Egr1 level was increased with increased Cav3.2 mRNA level at 3 days after TAB. To demonstrate that Egr1 indeed regulates Cav3.2 expression after hypertrophic stimulation, knockdown of Egr1 with short hairpin RNA prevented the phenylephrine-induced up-regulation of Cav3.2 expression and cellular hypertrophy in neonatal rat ventricular myocytes (NRVMs) and H9c2 cells. Furthermore, overexpression of Cav3.2 in Egr1-knockdown cells restored the phenylephrine-induced hypertrophy. Conclusion Cav3.2 is induced early by Egr1 during cardiac hypertrophy and Cav3.2 is an important mediator of Egr1 in regulating cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Highlights: [•] Cav3.2 interacts with calcineurin by an NFAT-like binding. [•] The Cav3.2/calcineurin interaction is dependent on Ca2+ and calmodulin. [•] The binding has inhibitory effects on both calcineurin and Cav3.2 activity. [•] We report a mechanism which prevents calcineurin from being activated by systolic Ca2+. [ABSTRACT FROM AUTHOR]

Aims Restenosis is an undesirable consequence following percutaneous vascular interventions. However, the current strategy for preventing restenosis is inadequate. The aim of this study was to investigate the role of low-voltage gated T-type calcium channels in regulating vascular smooth muscle cell (VSMC) proliferation during neointimal formation. Methods and results Wire injury of mice carotid arteries resulted in neointimal formation in the wild-type and Cav3.2−/− but not Cav3.1−/− mice, indicating a critical role of Cav3.1 in neointimal formation. In addition, we found a significant increase of Cav3.1 mRNA and protein in injured arteries. Cav3.1 knockout or knockdown (shCav3.1) reduced VSMC proliferation. Since T-channels are expressed predominantly in the G1 and S phases in VSMCs, we examined whether an abnormal G1/S transition was the cause of the reduced cell proliferation in shCav3.1 VSMCs. We found a disrupted expression of cyclin E in shCav3.1 VSMCs, and calmodulin agonist CALP1 partially rescued the defective cell proliferation. Furthermore, we demonstrated that infusion of NNC55-0396, a selective T-channel blocker, inhibited neointimal formation in wild-type mice. Conclusion Cav3.1 is required for VSMC proliferation during neointimal formation, and blocking of Cav3.1 may be beneficial for preventing restenosis. [ABSTRACT FROM PUBLISHER]

We characterized the neuronal two-domain (95kD-α12.1) form of the α12.1 subunit of the voltage-gated calcium channels using genetic and molecular analysis. The 95kD-α12.1 is absent in neuronal preparations from CACNA1A null mouse demonstrating that α12.1 and 95kD-α12.1 arise from the same gene. A recombinant two-domain form (α1AI-II) of α12.1 associates with the β subunit and is trafficked to the plasma membrane. Translocation of the α1AI-II to the plasma membrane requires association with the β subunit, since a mutation in the α1AI-II that inhibits β subunit association reduces membrane trafficking. Though the α1AI-II protein does not conduct any voltage-gated currents, we have previously shown that it generates a high density of non-linear charge movements [Ahern et al., Proc. Natl. Acad. Sci. USA 98 (2001) 6935–6940]. In this study, we demonstrate that co-expression of the α1AI-II significantly reduces the current amplitude of α12.1/β1a/α2δ channels, via competition for the β subunit. Taken together, our results demonstrate a dual functional role for the α1AI-II protein, both as a voltage sensor and modulator of P/Q-type currents in recombinant systems. These studies suggest an in vivo role for the 95kD-α12.1 in altering synaptic activity via protein–protein interactions and/or regulation of P/Q-type currents. [Copyright &y& Elsevier]

Calcium ion (Ca[sup2+]) influx through voltage-gated Ca[sup2+] channels is important for the regulation of vascular tone. Activation of L-type Ca[sup2+] channels initiates muscle contraction; however, the role of T-type Ca[sup2-] channels (T-channels) is not clear. We show that mice deficient in the α[supTH] T-type Ca[sup2+] channel (α,3.2-null) have constitutively constricted coronary arterioles and focal myocardial fibrosis. Coronary arteries isolated from α[sub1]3.2-null arteries showed normal contractile responses, but reduced relaxation in response to acetylcholine and nitroprusside. Furthermore, acute blockade of T-channels with Ni[sup2+] prevented relaxation of wild-type coronary arteries. Thus, Ca[sup2+] influx through α[sub1H] T-type Ca[sup2+] channels is essential for normal relaxation of coronary arteries. [ABSTRACT FROM AUTHOR]

Calcium channel β subunits expressed in rat atria and atrial myocytes are identified and their expression quantified and compared to β subunit expression in the ventricle. mRNAs encoding all four know β subunits are expressed in atrial myocytes including the following splice variants: β1a, β2b, β2c, β2e and β4d. The specific β2 splice variants expressed in the atria (β2b, β2c, β2e) differ from those previously reported from rat ventricle. β2 isoform is the most abundant β mRNA expressed in the heart and the amount of both β2 subunit mRNA and β2 subunit protein is significantly greater in the ventricles than in the atria. The expression of individual β2 splice variants varies with age and within different chambers of the heart. The β2b splice variant appears in both atria and ventricle in both young (4.5 week) and old (16 week) animals, the β2c isoform is more highly expressed in young as compared to old animals and the β2e splice variant is robustly expressed only in 4.5 week ventricle. β4 mRNA expression is higher in atrial tissue than in ventricles and its expression decreases in older animals. In contrast, the abundance of the β3 mRNA does not significantly change as a function of postnatal age. Antisense oligonucleotides targeting sequence common to all the β isoforms as well as that specific for β2 significantly reduced HVA calcium current density in isolated atrial cells confirming that the β2 subunits contribute to the regulation of HVA calcium current expression in the rat atria. The complexity of β isoform expression within the heart may provide a mechanism for functional fine-tuning of the cardiac HVA current. [Copyright &y& Elsevier]

Abstract: Loss of spiral ganglion neurons is a major cause of age-related hearing loss (presbycusis). Despite being the third most prevalent condition afflicting elderly persons, there are no known medications to prevent presbycusis. Because calcium signaling has long been implicated in age-related neuronal death, we investigated T-type calcium channels. This family is comprised of three members (Cav3.1, Cav3.2, and Cav3.3), based on their respective main pore-forming alpha subunits: α1G, α1H, and α1I. In the present study, we report a significant delay of age-related loss of cochlear function and preservation of spiral ganglion neurons in α1H null and heterozygous mice, clearly demonstrating an important role for Cav3.2 in age-related neuronal loss. Furthermore, we show that anticonvulsant drugs from a family of T-type calcium channel blockers can significantly preserve spiral ganglion neurons during aging. To our knowledge, this is the first report of drugs capable of diminishing age-related loss of spiral ganglion neurons. [Copyright &y& Elsevier]