L-type Ca2+ channels in Ca2+ channelopathies Striessnig, Jörg; Hoda, Jean-Charles; Koschak, Alexandra ...
Biochemical and biophysical research communications,
2004-Oct-01, 2004-10-00, Letnik:
322, Številka:
4
Journal Article
Recenzirano
Voltage-gated L-type Ca2+ channels (LTCCs) mediate depolarization-induced Ca2+ entry in electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells. In this review ...we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within pore-forming alpha1 subunits causing incomplete congenital stationary night blindness, malignant hyperthermia sensitivity or hypokalemic periodic paralysis. However, studies in mice revealed that LTCC dysfunction also contributes to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting molecular tools to elucidate the contribution of different LTCC isoforms to human diseases.
Immunological screening of a Pseudomonas aeruginosa cosmid library led to the identification of clones producing an 18 kDa outer-membrane protein. This protein reacted in Western blots with a ...polyclonal antiserum against outer-membrane proteins of P. aeruginosa and with a monoclonal antibody (MA1-6) specific for OprL, the peptidoglycan-associated outer-membrane lipoprotein (PAL). Sequencing of pOML7, a subclone expressing oprL, revealed an ORF of 504 bp encoding a polypeptide with a typical lipoprotein signal recognition sequence. Another ORF was found upstream of oprL, with homology to the TolB protein of Escherichia coli and Haemophilus influenzae. Downstream of oprL, a second ORF, of 321 bp, was found (orf2), encoding a protein with a signal peptide and with no homology with proteins of known biological function. After the stop codon of orf2, a rho-independent terminator sequence was detected which is part of the P. aeruginosa PAO1 insertion element IS222. OprL showed homologies with all known PALs from Gram-negative bacteria, especially in the C-terminal part. mAb MA1-6 reacted with P. aeruginosa cells in immunofluorescence, and with E. coli cells expressing oprL, which had an abnormal, elongated morphology, an indication that production of the protein perturbed the division process.
Replacement of L-type Ca2+ channel α1 subunit residue Thr-1066 in segment IIIS5 by a tyrosine residue conserved in the corresponding positions of non-L-type Ca2+ channels eliminates high ...dihydropyridine sensitivity through a steric mechanism. To determine the effects of this mutation on phenylalkylamine interaction, we exploited the availability of Cav1.2DHP–/– mice containing the T1066Y mutation. In contrast to dihydropyridines, increased protein-dependent binding of the phenylalkylamine (–)-3Hdevapamil occurred to Cav1.2DHP–/– mouse brain microsomes. This effect could be attributed to an at least 2-fold increase in affinity as determined by saturation analysis and binding inhibition experiments. The latter also revealed a higher affinity for (–)-verapamil but not for (–)-gallopamil. The mutation caused a pronounced slowing of (–)-3Hdevapamil dissociation, indicating a stabilization of the drug-channel complex. The increased affinity of mutant channels was also evident in functional studies after heterologous expression of wild type and T1066Y channels in Xenopus laevis oocytes. 100 μm (–)-verapamil inhibited a significantly larger fraction of Ba2+ inward current through mutant than through WT channels. Our results provide evidence that phenylalkylamines also interact with the IIIS5 helix and that the geometry of the IIIS5 helix affects the access and/or binding of different chemical classes of Ca2+ channel blockers to their overlapping binding domains. Mutation of Thr-1066 to a non-L-type tyrosine residue can be exploited to differentially affect phenylalkylamine and dihydropyridine binding to L-type Ca2+ channels.
Ca sub(V)1.2 and Ca sub(V)1.3, are the main dihydropyridine-sensitive L-type calcium channel isoforms in the brain. To reveal the contribution of each isoform to the neuronal activation pattern ...elicited by the dihydropyridine L- type calcium channel activator BayK 8644, we utilized Fos expression as a marker of neuronal activation in mutant mice (Ca sub(V)1.2 super(DHP-/-) mice) expressing dihydropyridine-insensitive Ca sub(V)1.2 L-type calcium channels. BayK 8644- treated wildtype mice displayed intense and widespread Fos expression throughout the neuroaxis in 77 of 80 brain regions quantified. The Fos response in Ca sub(V)1.2 super(DHP-/-) mice was greatly attenuated or absent in most of these areas, suggesting that a major part of the widespread Fos induction including most cortical areas was mediated by Ca sub(V)1.2 L-type calcium channels. BayK 8644-induced Fos expression in Ca sub(V)1.2 super(DHP-/-) mice indicating predominantly Ca sub(V)1.3 L-type calcium channel-mediated activation was noted in more restricted neuronal populations (20 of 80), in particular in the central amygdala, the bed nucleus of the stria terminalis, paraventricular hypothalamic nucleus, lateral preoptic area, locus coeruleus, lateral parabrachial nucleus, central nucleus of the inferior colliculus, and nucleus of the solitary tract. Our data indicate that selective stimulation of other than Ca sub(V)1.2 L-type calcium channels, mostly Ca sub(V)1.3, causes neuronal activation in a specific set of mainly limbic, hypothalamic and brainstem areas, which are associated with functions including integration of emotion-related behavior. Hence, selective modulation of Ca sub(V)1.3 L-type calcium channels could represent a novel (pharmacotherapeutic) tool to influence these CNS functions.
We report highly tunable control of holes in Ge/Si core/shell nanowires (NWs). We demonstrate the ability to create single quantum dots (QDs) of various sizes, with low hole occupation numbers and ...clearly observable excited states. For the smallest dot size we observe indications of single-hole occupation. Moreover, we create double and triple tunnel-coupled quantum dot arrays. In the double quantum dot configuration we observe Pauli spin blockade (PSB). These results open the way to perform hole spin qubit experiments in these devices.
We evaluated the possible interdependency of α2 Na+ pumps, Na+/Ca2+ exchanger (NCX), store‐operated channels (SOC) and L‐type voltage‐gated Ca2+ channels (LVC) in the control of sarcoplasmic ...reticulum (SR) Ca2+ release and myogenic tone (MT) in pressurized mouse mesenteric small arteries. To define the role of LVC, we used mice in which the Cav1.2 LVC are resistant to dihydropyridines (DHPR/R). Nifedipine (Nif, 0.3 – 1 μM) abolished high K+‐induced contraction and MT in wild type (WT) but not DHPR/R arteries. Thus Ca2+ entry via Cav1.2 LVC is the major pathway for maintaining MT and ≤ 1 μM Nif inhibits MT only by blocking LVC. In WT arteries, after SR depletion and MT abolition, readmission of external Ca2+ induced MT recovery. This involved activation of LVC because it was abolished by Nif pretreatment. MT recovery was partially blocked and slowed by SEA0400 (0.3 – 1 μM, a selective NCX inhibitor), and SK&F‐96365 (3–10 μM, a SOC inhibitor). This implies that the activity of DHP‐sensitive LVC depends on SOC and NCX. SR Ca2+ stores, measured by phenylephrine (5 μM) in Ca2+‐free external solution were only partially refilled upon restoration of external Ca2+. Increased SR Ca2+ release was observed in both α2 Na+ pump heterozygotes and NCX heterozygotes. These results suggest a model of Ca2+ dynamics in which SR refilling and MT generation depend on SOC, NCX and LVC. LVC may be activated by depolarization produced by cation entry through SOC and by Ca2+ influx through NCX. Furthermore, NCX and α2 Na+ pumps control SR Ca2+ content.
Supported by NHLBI and Austrian Science Funds (P‐17159).
To combine conformity of the irradiation with time effectiveness during treatment, the use of multileaf collimators has become more and more common. However, the simulation of the leaf positions is ...rather difficult compared with metal blocks. We developed a new method, utilizing an acrylic template in which the contour produced by the leaves is machined in the form of a 1 mm groove by a computerized numerically controlled milling machine. This template is then inserted into a mount attached to the simulator. The main advantages are the errorfree, direct communication from the therapy planning system to the milling machine via a network, the possibility to transfer the contour to the skin, and the documentation on the simulation film. The use of templates is reliable and, e.g. the costs of the materials are lower than for block simulation.
Ca sub(v)1.2 and Ca sub(v)1.3 L-type Ca super(2+) channels (LTCCs) are believed to underlie Ca super(2+) currents in brain, pancreatic beta cells, and the cardiovascular system. In the CNS, neuronal ...LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause card iovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca sub(v)1.2 alpha 1 subunits (Ca sub(v)1.2DHP-/-) without affecting function and expression. This allowed separation of the DHP effects of Ca sub(v)1.2 from those of Ca sub(v)1.3 and other LTCCs. DHP effects on pancreatic beta cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Ca sub(v)1.2DHP-/- mice, which rules out a direct role of Ca sub(v)1.3 for these physiological processes. Using Ca sub(v)1.2DHP-/- mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Ca sub(v)1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.
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