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1.
J Nanobiotechnology ; 21(1): 60, 2023 Feb 22.
Article in English | MEDLINE | ID: mdl-36814238

ABSTRACT

BACKGROUND: Unlike the injectable vaccines, intranasal lipid nanoparticle (NP)-based adjuvanted vaccine is promising to protect against local infection and viral transmission. Infection of ferrets with SARS-CoV-2 results in typical respiratory disease and pathology akin to in humans, suggesting that the ferret model may be ideal for intranasal vaccine studies. RESULTS: We developed SARS-CoV-2 subunit vaccine containing both Spike receptor binding domain (S-RBD) and Nucleocapsid (N) proteins (NP-COVID-Proteins) or their mRNA (NP-COVID-mRNA) and NP-monosodium urate adjuvant. Both the candidate vaccines in intranasal vaccinated aged ferrets substantially reduced the replicating virus in the entire respiratory tract. Specifically, the NP-COVID-Proteins vaccine did relatively better in clearing the virus from the nasal passage early post challenge infection. The immune gene expression in NP-COVID-Proteins vaccinates indicated increased levels of mRNA of IFNα, MCP1 and IL-4 in lungs and nasal turbinates, and IFNγ and IL-2 in lungs; while proinflammatory mediators IL-1ß and IL-8 mRNA levels in lungs were downregulated. In NP-COVID-Proteins vaccinated ferrets S-RBD and N protein specific IgG antibodies in the serum were substantially increased at both day post challenge (DPC) 7 and DPC 14, while the virus neutralizing antibody titers were relatively better induced by mRNA versus the proteins-based vaccine. In conclusion, intranasal NP-COVID-Proteins vaccine induced balanced Th1 and Th2 immune responses in the respiratory tract, while NP-COVID-mRNA vaccine primarily elicited antibody responses. CONCLUSIONS: Intranasal NP-COVID-Proteins vaccine may be an ideal candidate to elicit increased breadth of immunity against SARS-CoV-2 variants.


Subject(s)
COVID-19 , Influenza Vaccines , Humans , Animals , Aged , Ferrets , Immunity, Mucosal , SARS-CoV-2 , Viral Load , Antibodies, Viral , Lung/pathology , Antibodies, Neutralizing , Adjuvants, Immunologic , COVID-19 Vaccines , mRNA Vaccines
2.
Nat Commun ; 4: 1837, 2013.
Article in English | MEDLINE | ID: mdl-23673622

ABSTRACT

Calcium fluxes have been implicated in the specification of the vertebrate embryonic nervous system for some time, but how these fluxes are regulated and how they relate to the rest of the neural induction cascade is unknown. Here we describe Calfacilitin, a transmembrane calcium channel facilitator that increases calcium flux by generating a larger window current and slowing inactivation of the L-type CaV1.2 channel. Calfacilitin binds to this channel and is co-expressed with it in the embryo. Regulation of intracellular calcium by Calfacilitin is required for expression of the neural plate specifiers Geminin and Sox2 and for neural plate formation. Loss-of-function of Calfacilitin can be rescued by ionomycin, which increases intracellular calcium. Our results elucidate the role of calcium fluxes in early neural development and uncover a new factor in the modulation of calcium signalling.


Subject(s)
Calcium Channels/metabolism , Membrane Proteins/metabolism , Neural Plate/embryology , Neural Plate/metabolism , Animals , Body Patterning/drug effects , Body Patterning/genetics , Calcium/metabolism , Calcium Channels, L-Type/metabolism , Calcium Signaling/drug effects , Calcium Signaling/genetics , Cell Membrane/drug effects , Cell Membrane/metabolism , Chick Embryo , Geminin/metabolism , Gene Expression Regulation, Developmental/drug effects , Germ Layers/cytology , Germ Layers/drug effects , Germ Layers/metabolism , HEK293 Cells , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Membrane Proteins/genetics , Molecular Sequence Data , Morpholinos/pharmacology , Neural Plate/drug effects , Quail
3.
J Neurophysiol ; 103(6): 3311-9, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20393063

ABSTRACT

The Kv1.3 voltage-dependent potassium channel is expressed at high levels in mitral cells of the olfactory bulb (OB). Deletion of the Kv1.3 potassium channel gene (Kv1.3-/-) in mice lowers the threshold for detection of odors, increases the ability to discriminate between odors, and alters the firing pattern of mitral cells. We have now found that loss of Kv1.3 produces a compensatory increase in Na(+)-activated K(+) currents (K(Na)) in mitral cells. Levels of the K(Na) channel subunit Slack-B determined by Western blotting are substantially increased in the OB from Kv1.3-/- animals compared with those of wildtype animals. In voltage-clamp recordings of OB slices, elevation of intracellular sodium from 0 to 60 mM increased mean outward currents by 15% in mitral cells from wildtype animals and by 40% in cells from Kv1.3-/- animals. In Kv1.3-/- cells, K(Na) current could even be detected with 0 mM Na(+) internal solutions, provided extracellular Na(+) was present, and this current could be abolished by TTX and ZD7288, blockers of Na(+) influx through voltage-dependent Na(+) channels and H-channels, respectively. The role of enhanced expression of Slack subunits in the increase of K(Na) current in Kv1.3-/- cells was also confirmed using an RNA interference (RNA(i)) approach to suppress Slack expression in primary cultures of olfactory neurons. In Kv1.3-/- neurons, treatment with Slack-specific RNA(i) inhibited approximately 75% of the net outward current, whereas in wildtype cells, the same treatment suppressed only about 25% of the total current. Scrambled and mismatched RNA(i) oligonucleotides failed to suppress currents. Our findings raise the possibility that the olfactory phenotype of Kv1.3-/- animals results in part from an enhancement of K(Na) currents.


Subject(s)
Gene Expression Regulation/genetics , Kv1.3 Potassium Channel/deficiency , Neurons/physiology , Olfactory Bulb/cytology , Potassium Channels/metabolism , Animals , Animals, Newborn , Biophysics/methods , Cardiovascular Agents/pharmacology , Cells, Cultured , Electric Stimulation/methods , In Vitro Techniques , Membrane Potentials/genetics , Membrane Potentials/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins , Patch-Clamp Techniques/methods , Potassium Channels/genetics , Potassium Channels, Sodium-Activated , Pyrimidines/pharmacology , RNA Interference/physiology , Sodium Channel Blockers/pharmacology , Tetrodotoxin/pharmacology , Transfection/methods
4.
Pflugers Arch ; 456(6): 1085-95, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18369661

ABSTRACT

Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel Na(V)1.4 expressed in mammalian cells was studied by applying the Met-preferring oxidant chloramine-T or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of Met1470 in the putative receptor of the inactivation lid also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues in intracellular linkers connecting the membrane-spanning segments (442, 1139, 1154, 1316, 1469) were of minor importance. The results of mutagenesis, assays of other Na(V) channel isoforms (Na(V)1.2, Na(V)1.5, Na(V)1.7), and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of Na(V) channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion.


Subject(s)
Methionine/metabolism , Sodium Channel Blockers/pharmacology , Sodium Channels/drug effects , Sodium Channels/physiology , Algorithms , Cells, Cultured , Chloramines/pharmacology , Data Interpretation, Statistical , Electrophysiology , Fluorescent Dyes/pharmacology , Humans , Isoquinolines/pharmacology , Kinetics , Muscle Proteins/antagonists & inhibitors , Muscle Proteins/metabolism , Mutagenesis , NAV1.4 Voltage-Gated Sodium Channel , Oxidation-Reduction , Patch-Clamp Techniques , Plasmids/genetics , Sodium Channels/genetics , Sodium Channels/metabolism , Tosyl Compounds/pharmacology
5.
Biochim Biophys Acta ; 1783(1): 118-30, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18070605

ABSTRACT

Cav1.2 L-type calcium channels are essential in heart and smooth muscle contraction. Rat Cav1.2 gene contains 11 alternatively spliced exons (1a, 1, 8a, 8, 9*, 21, 22, 31, 32, 32-6nt and 33) which can be assorted to generate a large number of functionally distinct splice variants. Until now, it is unknown whether the utilization of these alternatively spliced exons is altered in the hypertrophied hearts of hypertensive rats. By comparing the assortments of these 11 exons in full-length Cav1.2 transcripts derived from Spontaneously Hypertensive Rats (SHRs) and Wistar Kyoto Rats (WKYs) hearts, we found that the inclusion of Cav1.2 alternative exons was significantly different between the two rats both at individual loci and in combinatorial arrangements. Functional characterizations of three Cav1.2 channel splice variants that were identified to be significantly altered in SHR hypertrophied cardiomyocytes demonstrated distinct whole-cell electrophysiological properties when expressed in HEK 293 cells. Interestingly, aberrant splice variants which included or excluded both mutually exclusive exons 21/22 or exons 31/32 were found to be increased in hypertensive rats. Two aberrant splice variants that included both exons 21 and 22 were found to be unable to conduct currents even though they expressed proteins with the predicted molecular mass. Characterization of one of the aberrant splice variants showed that it exerted a dominant negative effect on the functional Cav1.2 channels when co-expressed in HEK293 cells. The altered combinatorial splicing profiles of Cav1.2 transcripts identified in SHR hearts provide a different and new perspective in understanding the possible role of molecular remodeling of Cav1.2 channels in cardiac hypertrophy as a consequence of hypertension.


Subject(s)
Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/metabolism , Myocardium/metabolism , RNA Splicing/genetics , Animals , Blood Pressure , Cell Line , DNA, Complementary/genetics , Electrophysiology , Exons/genetics , Gene Expression Regulation , Humans , Hypertension/genetics , Hypertension/metabolism , Hypertension/physiopathology , Organ Size , Patch-Clamp Techniques , Protein Subunits/genetics , Protein Subunits/metabolism , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Transcription, Genetic/genetics
6.
J Biol Chem ; 279(48): 50329-35, 2004 Nov 26.
Article in English | MEDLINE | ID: mdl-15381693

ABSTRACT

Voltage-gated calcium channels play a major role in many important processes including muscle contraction, neurotransmission, excitation-transcription coupling, and hormone secretion. To date, 10 calcium channel alpha(1)-subunits have been reported, of which four code for L-type calcium channels. In our previous work, we uncovered by transcript-scanning the presence of 19 alternatively spliced exons in the L-type Ca(v)1.2 alpha(1)-subunit. Here, we report the smooth muscle-selective expression of alternatively spliced exon 9(*) in Ca(v)1.2 channels found on arterial smooth muscle. Specific polyclonal antibody against exon 9(*) localized the intense expression of 9(*)-containing Ca(v)1.2 channels on the smooth muscle wall of arteries, but the expression on cardiac muscle was low. Whole-cell patch clamp recordings of the 9(*)-containing Ca(v)1.2 channels in HEK293 cells demonstrated -9 and -11-mV hyperpolarized shift in voltage-dependent activation and current-voltage relationships, respectively. The steady-state inactivation property and sensitivity to blockade by nifedipine of the +/-exon 9(*) splice variants were, however, not significantly different. Such cell-selective expression of an alternatively spliced exon strongly indicates the customization and fine tuning of calcium channel functions through alternative splicing of the pore-forming alpha(1)-subunit. The generation of proteomic variations by alternative splicing of the calcium channel Ca(v)1.2 alpha(1)-subunit can potentially provide a flexible mechanism for muscle or neuronal cells to respond to various physiological signals or to diseases.


Subject(s)
Alternative Splicing , Calcium Channels, L-Type/genetics , Muscle, Smooth/metabolism , Amino Acid Sequence , Antibodies , Blotting, Western , Calcium Channels, L-Type/biosynthesis , Calcium Channels, L-Type/immunology , Calcium Channels, L-Type/metabolism , Electrophysiology , Exons , Humans , Immunohistochemistry , Molecular Sequence Data
7.
J Biol Chem ; 279(43): 44335-43, 2004 Oct 22.
Article in English | MEDLINE | ID: mdl-15299022

ABSTRACT

The L-type (Cav1.2) voltage-gated calcium channels play critical roles in membrane excitability, gene expression, and muscle contraction. The generation of splice variants by the alternative splicing of the poreforming Cav1.2 alpha1-subunit (alpha(1)1.2) may thereby provide potent means to enrich functional diversity. To date, however, no comprehensive scan of alpha(1)1.2 splice variation has been performed, particularly in the human context. Here we have undertaken such a screen, exploiting recently developed "transcript scanning" methods to probe the human gene. The degree of variation turns out to be surprisingly large; 19 of the 55 exons comprising the human alpha(1)1.2 gene were subjected to alternative splicing. Two of these are previously unrecognized exons and two others were not known to be spliced. Comparisons of fetal and adult heart and brain uncovered a large IVS3-S4 variability resulting from combinatorial utilization of exons 31-33. Electrophysiological characterization of such IVS3-S4 variation revealed unmistakable shifts in the voltage dependence of activation, according to an interesting correlation between increased IVS3-S4 linker length and activation at more depolarized potentials. Steady-state inactivation profiles remained unaltered. This systematic portrait of splice variation furnishes a reference library for comprehending combinatorial arrangements of Cav1.2 splice exons, especially as they impact development, physiology, and disease.


Subject(s)
Calcium Channels, L-Type/biosynthesis , Calcium Channels, L-Type/genetics , RNA, Messenger/metabolism , Alternative Splicing , Amino Acid Sequence , Barium/chemistry , Brain/metabolism , Cell Line , DNA Primers/chemistry , DNA, Complementary/metabolism , Electrophysiology , Exons , Genetic Variation , Humans , Models, Biological , Molecular Sequence Data , Myocardium/metabolism , Patch-Clamp Techniques , Polymerase Chain Reaction , Protein Conformation , Protein Structure, Tertiary , RNA Splicing , Tissue Distribution
8.
Mol Pharmacol ; 65(3): 685-91, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14978247

ABSTRACT

Scorpion alpha-toxins LqhalphaIT, Lqh-2, and Lqh-3 are representatives of three groups of alpha-toxins that differ in their preference for insects and mammals. These alpha-insect, antimammalian, and alpha-like toxins bind to voltage-gated sodium channels and slow down channel inactivation. Sodium channel mutagenesis studies using various alpha-toxins have shown that they interact with receptor site 3, which is composed mainly of a short stretch of amino-acid residues between S3 and S4 of domain 4. Variation in this region results in marked differences between various subtypes of sodium channels with respect to their sensitivity to the three Lqh toxins. We incorporated the S3-S4 linker of domain 4 from hNaV1.2/hNaV1.1, hNaV1.3, hNaV1.6, and hNaV1.7 channels as well as individual point mutations into the rNaV1.4 skeletal muscle sodium channel. Our data show that the affinity of Lqh-3 and LqhalphaIT to sodium channels is markedly determined by an aspartate residue (Asp1428 in rNaV1.4); when mutated to glutamate, as is present in NaV1.1-1.3 channels, Lqh-3-channel interactions are abolished. The interaction of Lqh-2 and LqhalphaIT, however, is strongly reduced when a lysine residue (Lys1432 in rNaV1.4) is replaced by threonine (as in hNaV1.7), whereas this substitution is without effect for Lqh-3. The influence of Lys1432 on Lqh-2 and LqhalphaIT strongly depends on the context of the Asp/Glu site at position 1428, giving rise to a wide variety of toxicological phenotypes by means of a combinatorial mixing and matching of only a few residues in receptor site 3.


Subject(s)
Muscle Proteins/metabolism , Nerve Tissue Proteins/metabolism , Scorpion Venoms/toxicity , Sodium Channels/metabolism , Animals , Binding Sites , Cells, Cultured , Humans , Muscle Proteins/drug effects , Muscle Proteins/genetics , Mutagenesis, Site-Directed , NAV1.1 Voltage-Gated Sodium Channel , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/genetics , Rats , Scorpion Venoms/chemistry , Sodium Channels/drug effects , Sodium Channels/genetics , Transfection
9.
Eur J Neurosci ; 16(4): 767-70, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12270053

ABSTRACT

The scorpion alpha-toxins Lqh-2 and Lqh-3, isolated from the venom of the Israeli yellow scorpion Leiurus quinquestriatus hebraeus, were previously shown to be very potent in removing fast inactivation of rat skeletal muscle sodium channels (Chen et al., 2000). Here, we show that tetrodotoxin-sensitive neuronal channels NaV1.2 and NaV1.7, which are mainly expressed in mammalian central and peripheral nervous systems, respectively, are differentially sensitive to these two toxins. rNaV1.2 and hNaV1.7 channels were studied with patch-clamp methods upon expression in mammalian cells. While Lqh-3 was about 100-times more potent in removing inactivation in hNaV1.7 channels compared with rNaV1.2, Lqh-2 was about 20-times more active in the other direction. Site-directed mutagenesis showed that the differences in the putative binding sites for these toxins, the S3-4 linkers of domain 4, are of major importance for Lqh-3, but not for Lqh-2.


Subject(s)
Central Nervous System/drug effects , Peripheral Nervous System/drug effects , Scorpion Venoms/pharmacology , Sodium Channels/physiology , Animals , Central Nervous System/physiology , Humans , NAV1.2 Voltage-Gated Sodium Channel , Nerve Tissue Proteins/physiology , Peripheral Nervous System/physiology
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