The translocation of a chloride channel from the Golgi to the plasma membrane helps plants adapt to salt stress.

A key mechanism employed by plants to adapt to salinity stress involves maintaining ion homeostasis via the actions of ion transporters. While the function of cation transporters in maintaining ion homeostasis in plants has been extensively studied, little is known about the roles of their anion counterparts in this process. Here, we describe a mechanism of salt adaptation in plants. We characterized the chloride channel (CLC) gene AtCLCf, whose expression is regulated by WRKY transcription factor under salt stress in Arabidopsis thaliana. Loss-of-function atclcf seedlings show increased sensitivity to salt, whereas AtCLCf overexpression confers enhanced resistance to salt stress. Salt stress induces the translocation of GFP-AtCLCf fusion protein to the plasma membrane (PM). Blocking AtCLCf translocation using the exocytosis inhibitor brefeldin-A or mutating the small GTPase gene AtRABA1b/BEX5 (RAS GENES FROM RAT BRAINA1b homolog) increases salt sensitivity in plants. Electrophysiology and liposome-based assays confirm the Cl-/H+ antiport function of AtCLCf. Therefore, we have uncovered a mechanism of plant adaptation to salt stress involving the NaCl-induced translocation of AtCLCf to the PM, thus facilitating Cl- removal at the roots, and increasing the plant's salinity tolerance.

MFSD7c functions as a transporter of choline at the blood-brain barrier.

Mutations in the orphan transporter MFSD7c (also known as Flvcr2), are linked to Fowler syndrome. Here, we used Mfsd7c knockout (Mfsd7c-/-) mice and cell-based assays to reveal that MFSD7c is a choline transporter at the blood-brain barrier (BBB). We performed comprehensive metabolomics analysis and detected differential changes of metabolites in the brains and livers of Mfsd7c-/-embryos. Particularly, we found that choline-related metabolites were altered in the brains but not in the livers of Mfsd7c-/- embryos. Thus, we hypothesized that MFSD7c regulates the level of choline in the brain. Indeed, expression of human MFSD7c in cells significantly increased choline uptake. Interestingly, we showed that choline uptake by MFSD7c is greatly increased by choline-metabolizing enzymes, leading us to demonstrate that MFSD7c is a facilitative transporter of choline. Furthermore, single-cell patch clamp analysis showed that the import of choline by MFSD7c is electrogenic. Choline transport function of MFSD7c was shown to be conserved in vertebrates, but not in yeasts. We demonstrated that human MFSD7c is a functional ortholog of HNM1, the yeast choline importer. We also showed that several missense mutations identified in patients exhibiting Fowler syndrome had abolished or reduced choline transport activity. Mice lacking Mfsd7c in endothelial cells of the central nervous system suppressed the import of exogenous choline from blood but unexpectedly had increased choline levels in the brain. Stable-isotope tracing study revealed that MFSD7c was required for exporting choline derived from lysophosphatidylcholine in the brain. Collectively, our work identifies MFSD7c as a choline exporter at the BBB and provides a foundation for future work to reveal the disease mechanisms of Fowler syndrome.

Observation of fractal higher-order topological states in acoustic metamaterials.

Topological phases of matter have been extensively investigated in solid-state materials and classical wave systems with integer dimensions. However, topological states in non-integer dimensions remain almost unexplored. Fractals, being self-similar on different scales, are one of the intriguing complex geometries with non-integer dimensions. Here, we demonstrate fractal higher-order topological states with unprecedented emergent phenomena in a Sierpinski acoustic metamaterial. We uncover abundant topological edge and corner states in the acoustic metamaterial due to the fractal geometry. Interestingly, the numbers of the edge and corner states depend exponentially on the system size, and the leading exponent is the Hausdorff fractal dimension of the Sierpinski carpet. Furthermore, the results reveal the unconventional spectrum and rich wave patterns of the corner states with consistent simulations and experiments. This study thus unveils unconventional topological states in fractal geometry and may inspire future studies of topological phenomena in non-Euclidean geometries.

Muscle 4EBP1 activation modifies the structure and function of the neuromuscular junction in mice.

Dysregulation of mTOR complex 1 (mTORC1) activity drives neuromuscular junction (NMJ) structural instability during aging; however, downstream targets mediating this effect have not been elucidated. Here, we investigate the roles of two mTORC1 phosphorylation targets for mRNA translation, ribosome protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), in regulating NMJ structural instability induced by aging and sustained mTORC1 activation. While myofiber-specific deletion of S6k1 has no effect on NMJ structural integrity, 4EBP1 activation in murine muscle induces drastic morphological remodeling of the NMJ with enhancement of synaptic transmission. Mechanistically, structural modification of the NMJ is attributed to increased satellite cell activation and enhanced post-synaptic acetylcholine receptor (AChR) turnover upon 4EBP1 activation. Considering that loss of post-synaptic myonuclei and reduced NMJ turnover are features of aging, targeting 4EBP1 activation could induce NMJ renewal by expanding the pool of post-synaptic myonuclei as an alternative intervention to mitigate sarcopenia.

S-Nitrosylation-Mediated Reduction of CaV1.2 Surface Expression and Open Probability Underlies Attenuated Vasoconstriction Induced by Nitric Oxide.

BACKGROUND: L-type CaV1.2 calcium channel, the primary gateway for Ca2+ influx in smooth muscles, is widely regulated by multiple posttranslational modifications, such as protein kinase-mediated phosphorylation and nitric oxide-induced S-nitrosylation. However, the effect of S-nitrosylation on CaV1.2 channel function and its role in arterial contractility are not well understood. METHODS: Electrophysiological recordings, Ca2+ and confocal imaging, and biochemical assays were used to functionally characterize S-nitrosylated CaV1.2 channels in vitro, while pressure myography and tail-cuff blood pressure measurement were conducted to evaluate the physiological effects of CaV1.2 S-nitrosylation ex vivo and in vivo. RESULTS: S-nitrosylation significantly reduced the CaV1.2 current density by promoting lysosomal degradation that leads to decreased levels of total and surface CaV1.2 channel proteins in a CaVß-independent manner and reducing the open probability of CaV1.2 channel. Mechanistically, the Cys1180 and Cys1280 residues within CaV1.2 channel have been determined as the molecular targets for S-nitrosylation as substitution of either Cys1180 or Cys1280 for serine resulted in substantial reduction of S-nitrosylation levels. Of note, CaV1.2 S-nitrosylation levels were significantly reduced in arteries isolated from both spontaneously hypertensive rats and patients with pulmonary hypertension. Moreover, mouse resistance arteries incubated with S-nitrosocysteine displayed much lower contractility and spontaneously hypertensive rats injected with S-nitrosocysteine also showed significantly reduced blood pressure, suggesting that reduced S-nitrosylation contributes to the upregulation of CaV1.2 channel activity in hypertensive arteries. CONCLUSIONS: This study provides strong evidence that S-nitrosylation-mediated downregulation of CaV1.2 channels is via 2 distinctive mechanisms and the findings offer potential pathways for therapeutic inventions in hypertension.

Stackelberg-Theoretic Approach for Performance Improvement in Fuzzy Systems.

This paper investigates the robust control for dynamical systems subject to uncertainty. The uncertainty is assumed to be (possibly fast) time varying and bounded. The bound is unknown but lies within a prescribed fuzzy set (hence the fuzzy dynamical system). We propose an approach for the robust control design which is implemented in two steps. First, a class of robust controls is proposed based on tunable parameters. The proposed controls are deterministic and are not conventionally IF-THEN rules based. By the Lyapunov minimax approach, we prove that the proposed controls are able to guarantee deterministic system performance, namely, uniform boundedness and ultimate uniform boundedness. Second, optima seeking from the proposed controls is considered to improve fuzzy system performance. We formulate the optima-seeking problem as a two-player (one leader and one follower) Stackelberg game by developing two cost functions, each of which is in charge of one tunable parameter (i.e., the player). Each cost function consists of an average fuzzy system performance index and the associated player's control effort. We show that the solution of the optimal design problem (i.e., the optima of the tunable parameters), which is called the Stackelberg strategy, always exists and how to obtain the backwards-induction outcome is provided. Simulation results on the walking control of a biped robot model are presented for demonstration.

TRPM4-specific blocking antibody attenuates reperfusion injury in a rat model of stroke.

Reperfusion therapy is currently the gold standard treatment for acute ischemic stroke. However, reperfusion injuries such as oedema and haemorrhagic transformation largely limit the use of this potent treatment to a narrow time window. Recently, transient receptor potential melastatin 4 (TRPM4) channel has emerged as a potential target for vascular protection in stroke management. Non-specificity and side effects are major concerns for current TRPM4 blockers. The present study was undertaken to develop a novel TRPM4 blocker for stroke management. We report the generation of a TRPM4-specific antibody M4P which binds to a region close to the channel pore. M4P could inhibit TRPM4 current and downregulate TRPM4 surface expression, therefore prevent hypoxia-induced cell swelling. In the rat model of 3-h stroke reperfusion, application of M4P at 2 h after occlusion ameliorated reperfusion injury by improving blood-brain barrier integrity, and enhanced functional recovery. Our results demonstrate that TRPM4 blockade could attenuate reperfusion injury in stroke recanalization. When applied together with reperfusion treatments, TRPM4 blocking antibody has the potential to extend the therapeutic time window for acute ischemic stroke.

Manipulating energy migration within single lanthanide activator for switchable upconversion emissions towards bidirectional photoactivation.

Reliance on low tissue penetrating UV or visible light limits clinical applicability of phototherapy, necessitating use of deep tissue penetrating near-infrared (NIR) to visible light transducers like upconversion nanoparticles (UCNPs). While typical UCNPs produce multiple simultaneous emissions for unidirectional control of biological processes, programmable control requires orthogonal non-overlapping light emissions. These can be obtained through doping nanocrystals with multiple activator ions. However, this requires tedious synthesis and produces complicated multi-shell nanoparticles with a lack of control over emission profiles due to activator crosstalk. Herein, we explore cross-relaxation (CR), a non-radiative recombination pathway typically perceived as deleterious, to manipulate energy migration within the same lanthanide activator ion (Er3+) towards orthogonal red and green emissions, simply by adjusting excitation wavelength from 980 to 808 nm. These UCNPs allow programmable activation of two synergistic light-gated ion channels VChR1 and Jaws in the same cell to manipulate membrane polarization, demonstrated here for cardiac pacing.

Elastic Higher-Order Topological Insulator with Topologically Protected Corner States.

Topologically gapless edge states, characterized by topological invariants and Berry's phases of bulk energy bands, provide amazing techniques to robustly control the reflectionless propagation of electrons, photons, and phonons. Recently, a new family of topological phases, dictated by the bulk polarization, has been observed, leading to the discovery of the higher-order topological insulators (HOTIs). So far, the HOTIs have been demonstrated in mechanical and electromagnetic systems and electrical circuits with quantized quadrupole polarization and, more recently, have been experimentally realized in optical and acoustic systems. Here, we realize the higher-order topological states in a two-dimensional (2D) continuous elastic system. We experimentally observe the gapped one-dimensional (1D) edge states, the trivially gapped zero-dimensional (0D) corner states, and the topologically protected 0D corner states. Compared with the trivial corner modes, the topological ones, immunizing against defects, are robustly localized at the obtuse-angled but not the acute-angled corners. The topological shape-dependent corner states open a new route for the design of the topologically protected and reconfigurable 0D localized resonances and provide an excellent platform for the topological transformation of the elastic energy among 2D bulk, 1D edge, and 0D corner modes.

S-Nitrosylation of Divalent Metal Transporter 1 Enhances Iron Uptake to Mediate Loss of Dopaminergic Neurons and Motoric Deficit.

Elevated iron deposition has been reported in Parkinson's disease (PD). However, the route of iron uptake leading to high deposition in the substantia nigra is unresolved. Here, we show a mechanism in enhanced Fe2+ uptake via S-nitrosylation of divalent metal transporter 1 (DMT1). While DMT1 could be S-nitrosylated by exogenous nitric oxide donors, in human PD brains, endogenously S-nitrosylated DMT1 was detected in postmortem substantia nigra. Patch-clamp electrophysiological recordings and iron uptake assays confirmed increased Mn2+ or Fe2+ uptake through S-nitrosylated DMT1. We identified two major S-nitrosylation sites, C23 and C540, by mass spectrometry, and DMT1 C23A or C540A substitutions abolished nitric oxide (NO)-mediated DMT1 current increase. To evaluate in vivo significance, lipopolysaccharide (LPS) was stereotaxically injected into the substantia nigra of female and male mice to induce inflammation and production of NO. The intranigral LPS injection resulted in corresponding increase in Fe2+ deposition, JNK activation, dopaminergic neuronal loss and deficit in motoric activity, and these were rescued by the NO synthase inhibitor l-NAME or by the DMT1-selective blocker ebselen. Lentiviral knockdown of DMT1 abolished LPS-induced dopaminergic neuron loss.SIGNIFICANCE STATEMENT Neuroinflammation and high cytoplasmic Fe2+ levels have been implicated in the initiation and progression of neurodegenerative diseases. Here, we report the unexpected enhancement of the functional activity of transmembrane divalent metal transporter 1 (DMT1) by S-nitrosylation. We demonstrated that S-nitrosylation increased DMT1-mediated Fe2+ uptake, and two cysteines were identified by mass spectrometry to be the sites for S-nitrosylation and for enhanced iron uptake. One conceptual advance is that while DMT1 activity could be increased by external acidification because the gating of the DMT1 transporter is proton motive, we discovered that DMT1 activity could also be enhanced by S-nitrosylation. Significantly, lipopolysaccharide-induced nitric oxide (NO)-mediated neuronal death in the substantia nigra could be ameliorated by using l-NAME, a NO synthase inhibitor, or by ebselen, a DMT1-selective blocker.

Alternative Splicing at N Terminus and Domain I Modulates CaV1.2 Inactivation and Surface Expression.

The CaV1.2 L-type calcium channel is a key conduit for Ca2+ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its α1C pore-forming subunit is known to undergo extensive alternative splicing to produce many CaV1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human CaV1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with CaVß2a, displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V1/2inact of CaV1.2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V1/2act. However, the measured effects were ß-subunit-dependent when comparing CaVß2a with CaVß3 coexpression. Notably, calcium-dependent inactivation mediated by local Ca2+-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing CaV1.2 as compared to exon-1a-containing CaV1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with CaVß2a or CaVß3 subunit. This finding correlated well with a higher channel surface expression for the exon 1-CaV1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human CaV1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.

Regulation of Blood Pressure by Targeting CaV1.2-Galectin-1 Protein Interaction.

BACKGROUND: L-type CaV1.2 channels play crucial roles in the regulation of blood pressure. Galectin-1 (Gal-1) has been reported to bind to the I-II loop of CaV1.2 channels to reduce their current density. However, the mechanistic understanding for the downregulation of CaV1.2 channels by Gal-1 and whether Gal-1 plays a direct role in blood pressure regulation remain unclear. METHODS: In vitro experiments involving coimmunoprecipitation, Western blot, patch-clamp recordings, immunohistochemistry, and pressure myography were used to evaluate the molecular mechanisms by which Gal-1 downregulates CaV1.2 channel in transfected, human embryonic kidney 293 cells, smooth muscle cells, arteries from Lgasl1-/- mice, rat, and human patients. In vivo experiments involving the delivery of Tat-e9c peptide and AAV5-Gal-1 into rats were performed to investigate the effect of targeting CaV1.2-Gal-1 interaction on blood pressure monitored by tail-cuff or telemetry methods. RESULTS: Our study reveals that Gal-1 is a key regulator for proteasomal degradation of CaV1.2 channels. Gal-1 competed allosterically with the CaVß subunit for binding to the I-II loop of the CaV1.2 channel. This competitive disruption of CaVß binding led to CaV1.2 degradation by exposing the channels to polyubiquitination. It is notable that we demonstrated that the inverse relationship of reduced Gal-1 and increased CaV1.2 protein levels in arteries was associated with hypertension in hypertensive rats and patients, and Gal-1 deficiency induces higher blood pressure in mice because of the upregulated CaV1.2 protein level in arteries. To directly regulate blood pressure by targeting the CaV1.2-Gal-1 interaction, we administered Tat-e9c, a peptide that competed for binding of Gal-1 by a miniosmotic pump, and this specific disruption of CaV1.2-Gal-1 coupling increased smooth muscle CaV1.2 currents, induced larger arterial contraction, and caused hypertension in rats. In contrasting experiments, overexpression of Gal-1 in smooth muscle by a single bolus of AAV5-Gal-1 significantly reduced blood pressure in spontaneously hypertensive rats. CONCLUSIONS: We have defined molecularly that Gal-1 promotes CaV1.2 degradation by replacing CaVß and thereby exposing specific lysines for polyubiquitination and by masking I-II loop endoplasmic reticulum export signals. This mechanistic understanding provided the basis for targeting CaV1.2-Gal-1 interaction to demonstrate clearly the modulatory role that Gal-1 plays in regulating blood pressure, and offering a potential approach for therapeutic management of hypertension.

Characterization of CaV1.2 exon 33 heterozygous knockout mice and negative correlation between Rbfox1 and CaV1.2 exon 33 expressions in human heart failure.

Recently, we reported that homozygous deletion of alternative exon 33 of CaV1.2 calcium channel in the mouse resulted in ventricular arrhythmias arising from increased CaV1.2Δ33 ICaL current density in the cardiomyocytes. We wondered whether heterozygous deletion of exon 33 might produce cardiac phenotype in a dose-dependent manner, and whether the expression levels of RNA splicing factors known to regulate alternative splicing of exon 33 might change in human heart failure. Unexpectedly, we found that exon 33+/- cardiomyocytes showed similar CaV1.2 channel properties as wild-type cardiomyocyte, even though CaV1.2Δ33 channels exhibit a gain-in-function. In human hearts, we found that the mRNA level of splicing factor Rbfox1, but not Rbfox2, was downregulated in dilated cardiomyopathy, and CACNA1C mRNA level was dramatically decreased in the both of dilated and ischemic cardiomyopathy. These data imply Rbfox1 may be involved in the development of cardiomyopathies via regulating the alternative splicing of CaV1.2 exon 33. (149 words).

Exclusion of alternative exon 33 of CaV1.2 calcium channels in heart is proarrhythmogenic.

Alternative splicing changes the CaV1.2 calcium channel electrophysiological property, but the in vivo significance of such altered channel function is lacking. Structure-function studies of heterologously expressed CaV1.2 channels could not recapitulate channel function in the native milieu of the cardiomyocyte. To address this gap in knowledge, we investigated the role of alternative exon 33 of the CaV1.2 calcium channel in heart function. Exclusion of exon 33 in CaV1.2 channels has been reported to shift the activation potential -10.4 mV to the hyperpolarized direction, and increased expression of CaV1.2Δ33 channels was observed in rat myocardial infarcted hearts. However, how a change in CaV1.2 channel electrophysiological property, due to alternative splicing, might affect cardiac function in vivo is unknown. To address these questions, we generated mCacna1c exon 33-/--null mice. These mice contained CaV1.2Δ33 channels with a gain-of-function that included conduction of larger currents that reflects a shift in voltage dependence and a modest increase in single-channel open probability. This altered channel property underscored the development of ventricular arrhythmia, which is reflected in significantly more deaths of exon 33-/- mice from ß-adrenergic stimulation. In vivo telemetric recordings also confirmed increased frequencies in premature ventricular contractions, tachycardia, and lengthened QT interval. Taken together, the significant decrease or absence of exon 33-containing CaV1.2 channels is potentially proarrhythmic in the heart. Of clinical relevance, human ischemic and dilated cardiomyopathy hearts showed increased inclusion of exon 33. However, the possible role that inclusion of exon 33 in CaV1.2 channels may play in the pathogenesis of human heart failure remains unclear.

Homogenization-based interval analysis for structural-acoustic problem involving periodical composites and multi-scale uncertain-but-bounded parameters.

This paper presents a homogenization-based interval analysis method for the prediction of coupled structural-acoustic systems involving periodical composites and multi-scale uncertain-but-bounded parameters. In the structural-acoustic system, the macro plate structure is assumed to be composed of a periodically uniform microstructure. The equivalent macro material properties of the microstructure are computed using the homogenization method. By integrating the first-order Taylor expansion interval analysis method with the homogenization-based finite element method, a homogenization-based interval finite element method (HIFEM) is developed to solve a periodical composite structural-acoustic system with multi-scale uncertain-but-bounded parameters. The corresponding formulations of the HIFEM are deduced. A subinterval technique is also introduced into the HIFEM for higher accuracy. Numerical examples of a hexahedral box and an automobile passenger compartment are given to demonstrate the efficiency of the presented method for a periodical composite structural-acoustic system with multi-scale uncertain-but-bounded parameters.

Aberrant Splicing Promotes Proteasomal Degradation of L-type CaV1.2 Calcium Channels by Competitive Binding for CaVß Subunits in Cardiac Hypertrophy.

Decreased expression and activity of CaV1.2 calcium channels has been reported in pressure overload-induced cardiac hypertrophy and heart failure. However, the underlying mechanisms remain unknown. Here we identified in rodents a splice variant of CaV1.2 channel, named CaV1.2e21+22, that contained the pair of mutually exclusive exons 21 and 22. This variant was highly expressed in neonatal hearts. The abundance of this variant was gradually increased by 12.5-folds within 14 days of transverse aortic banding that induced cardiac hypertrophy in adult mouse hearts and was also elevated in left ventricles from patients with dilated cardiomyopathy. Although this variant did not conduct Ca2+ ions, it reduced the cell-surface expression of wild-type CaV1.2 channels and consequently decreased the whole-cell Ca2+ influx via the CaV1.2 channels. In addition, the CaV1.2e21+22 variant interacted with CaVß subunits significantly more than wild-type CaV1.2 channels, and competition of CaVß subunits by CaV1.2e21+22 consequently enhanced ubiquitination and subsequent proteasomal degradation of the wild-type CaV1.2 channels. Our findings show that the resurgence of a specific neonatal splice variant of CaV1.2 channels in adult heart under stress may contribute to heart failure.

Alternative splicing generates a novel truncated Cav1.2 channel in neonatal rat heart.

L-type Cav1.2 Ca(2+) channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Cav1.2 Ca(2+) channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Cav1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Cav1.233L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Cav1.2 channels, Cav1.233L channels reduced the current density and altered the electrophysiological properties of the wild type Cav1.2 channels. Interestingly, the truncated 3.5-domain Cav1.233L channels also yielded a dominant negative effect on Cav1.3 channels, but not on Cav3.2 channels, suggesting that Cavß subunits is required for Cav1.233L regulation. A biochemical study provided evidence that Cav1.233L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Cav1.233L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Cav1.2 channels. Moreover, the human Cav1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Cav1.2 channel. An electrophysiological study showed that human Cav1.233L channel is a functional channel but conducts Ca(2+) ions at a much lower level.

Modest CaV1.342-selective inhibition by compound 8 is ß-subunit dependent.

Two voltage-gated calcium channel subtypes-CaV1.2 and CaV1.3-underlie the major L-type Ca(2+) currents in the mammalian central nervous system. Owing to their high sequence homology, the two channel subtypes share similar pharmacological properties, and at high doses classic calcium channel blockers, such as dihydropyridines, phenylalkylamines and benzothiazepines, do not discriminate between the two channel subtypes. Recent progress in treating Parkinson's disease (PD) was marked by the discovery of synthetic compound 8, which was reported to be a highly selective inhibitor of the CaV1.3 L-type calcium channels (LTCC). However, despite a previously reported IC50 of ~24 µM, in our hands inhibition of the full-length CaV1.342 by compound 8 at 50 µM reaches a maximum of 45%. Moreover, we find that the selectivity of compound 8 towards CaV1.3 relative to CaV1.2B15 channels is greatly influenced by the ß-subunit type and its splice isoform variants.

TRPM4 inhibition promotes angiogenesis after ischemic stroke.

Transient receptor potential melastatin 4 (TRPM4) is a voltage-dependent, nonselective cation channel. Under pathological conditions, sustained activation of TRPM4 leads to oncotic cell death. Here, we report the upregulation of TRPM4 in vascular endothelium following hypoxia/ischemia in vitro and in vivo. In human umbilical vein endothelial cells, TRPM4 expression was increased at both the mRNA and protein levels following oxygen-glucose deprivation. Blocking TRPM4 with 9-phenanthrol greatly enhanced tube formation on Matrigel. In a rat permanent middle cerebral artery occlusion model, TRPM4 was upregulated in the vascular endothelium within the penumbra region after stroke. TRPM4 expression peaked 1 day post-occlusion and gradually decreased. In vivo siRNA-mediated TRPM4 silencing enhanced angiogenesis and improved capillary integrity. A twofold reduction in infarct volume and a substantial recovery of motor function were observed in animals receiving the siRNA treatment. Interestingly, the protective effect of TRPM4 suppression disappeared 5 days after stroke induction, indicating that TRPM4 upregulation is critical for cerebral damage during the acute phase of stroke. TRPM4 could be a potential therapeutic target for ischemic stroke.