scholarly journals Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable

2021 ◽  
Author(s):  
Jinping Lu ◽  
Ingo Dreyer ◽  
Miles Sasha Dickinson ◽  
Sabine Panzer ◽  
Dawid Jaslan ◽  
...  
Keyword(s):  
Vicia Faba ◽  
Ecological Niche ◽  
Pore Channel ◽  
Wild Type ◽  
Vacuole Membrane ◽  
Gene Functions ◽  
Voltage Dependent ◽  
Fire Action Potential ◽  
Plant Families ◽  
Sv Channel

To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but blocked by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues Glu457, Glu605 and Asp606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche.

scholarly journals Enhanced Abiotic Stress Tolerance of Vicia faba L. Plants Heterologously Expressing the PR10a Gene from Potato

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 173
Author(s):  
Abeer F. Desouky ◽  
Ahmed H. Ahmed ◽  
Hartmut Stützel ◽  
Hans-Jörg Jacobsen ◽  
Yi-Chen Pao ◽  
...  
Keyword(s):  
Salt Stress ◽  
Abiotic Stress ◽  
Stress Tolerance ◽  
Vicia Faba ◽  
Faba Bean ◽  
Abiotic Stress Tolerance ◽  
Wild Type ◽  
Stress Injury ◽  
Bean Plants ◽  
Vicia Faba L

Pathogenesis-related (PR) proteins are known to play relevant roles in plant defense against biotic and abiotic stresses. In the present study, we characterize the response of transgenic faba bean (Vicia faba L.) plants encoding a PR10a gene from potato (Solanum tuberosum L.) to salinity and drought. The transgene was under the mannopine synthetase (pMAS) promoter. PR10a-overexpressing faba bean plants showed better growth than the wild-type plants after 14 days of drought stress and 30 days of salt stress under hydroponic growth conditions. After removing the stress, the PR10a-plants returned to a normal state, while the wild-type plants could not be restored. Most importantly, there was no phenotypic difference between transgenic and non-transgenic faba bean plants under well-watered conditions. Evaluation of physiological parameters during salt stress showed lower Na+-content in the leaves of the transgenic plants, which would reduce the toxic effect. In addition, PR10a-plants were able to maintain vegetative growth and experienced fewer photosystem changes under both stresses and a lower level of osmotic stress injury under salt stress compared to wild-type plants. Taken together, our findings suggest that the PR10a gene from potato plays an important role in abiotic stress tolerance, probably by activation of stress-related physiological processes.

scholarly journals Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

2003 ◽  
Vol 122 (3) ◽  
pp. 295-306 ◽  
Author(s):  
Sonia Traverso ◽  
Laura Elia ◽  
Michael Pusch
Keyword(s):  
Chloride Channels ◽  
Bound State ◽  
Side Chain ◽  
Pore Channel ◽  
Kinetic Properties ◽  
Wild Type ◽  
High Affinity ◽  
Critical Residue ◽  
Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

scholarly journals Voltage-Dependent Calcium Channel CaV1.3 Subunits Regulate the Light Peak of the Electroretinogram

2007 ◽  
Vol 97 (5) ◽  
pp. 3731-3735 ◽  
Author(s):  
Jiang Wu ◽  
Alan D. Marmorstein ◽  
Jörg Striessnig ◽  
Neal S. Peachey
Keyword(s):  
Calcium Channel ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Rod Photoreceptor ◽  
Wild Type ◽  
Fast Oscillation ◽  
Light Peak ◽  
Voltage Dependent Calcium Channel ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels

In response to light, the mouse retinal pigment epithelium (RPE) generates a series of slow changes in potential that are referred to as the c-wave, fast oscillation (FO), and light peak (LP) of the electroretinogram (ERG). The LP is generated by a depolarization of the basolateral RPE plasma membrane by the activation of a calcium-sensitive chloride conductance. We have previously shown that the LP is reduced in both mice and rats by nimodipine, which blocks voltage-dependent calcium channels (VDCCs) and is abnormal in lethargic mice, carrying a null mutation in the calcium channel β4 subunit. To define the α1 subunit involved in this process, we examined mice lacking CaV1.3. In comparison with wild-type (WT) control littermates, LPs were reduced in CaV1.3−/− mice. This pattern matched closely with that previously noted in lethargic mice, confirming a role for VDCCs in regulating the signaling pathway that culminates in LP generation. These abnormalities do not reflect a defect in rod photoreceptor activity, which provides the input to the RPE to generate the c-wave, FO, and LP, because ERG a-waves were comparable in WT and CaV1.3−/− littermates. Our results identify CaV1.3 as the principal pore-forming subunit of VDCCs involved in stimulating the ERG LP.

scholarly journals Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels

2018 ◽  
Vol 115 (47) ◽  
pp. 12051-12056 ◽  
Author(s):  
Akil A. Puckerin ◽  
Donald D. Chang ◽  
Zunaira Shuja ◽  
Papiya Choudhury ◽  
Joachim Scholz ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Channel Blockers ◽  
Wild Type ◽  
Voltage Dependent ◽  
Somatosensory Neurons ◽  
Potential Applications ◽  
Voltage Dependent Calcium Channels ◽  
Potential Therapeutics

Genetically encoded inhibitors for voltage-dependent Ca2+ (CaV) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca2+ (CaV1/CaV2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary CaVβ-subunits. To investigate latent CaVβ-independent components of inhibition, we coexpressed each RGK individually with CaV1 (CaV1.2/CaV1.3) or CaV2 (CaV2.1/CaV2.2) channels reconstituted in HEK293 cells with either wild-type (WT) β2a or a mutant version (β2a,TM) that does not bind RGKs. All four RGKs strongly inhibited CaV1/CaV2 channels reconstituted with WT β2a. By contrast, when channels were reconstituted with β2a,TM, Rem inhibited only CaV1.2, Rad selectively inhibited CaV1.2 and CaV2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT CaVβ, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted CaV1.2 while Rad[R208A/L235A] inhibited CaV1.2/CaV2.2 but not CaV1.3/CaV2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous CaV1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for CaV1.2 and CaV1.2/CaV2.2 channels. Thus, we have exploited latent β-binding–independent Rem and Rad inhibition of specific CaV1/CaV2 channels to develop selective GECCIs with properties unmatched by current small-molecule CaV channel blockers.

Origami in the outer membrane: the transmembrane arrangement of mitochondrial porins

2002 ◽  
Vol 80 (5) ◽  
pp. 551-562 ◽  
Author(s):  
Denice C Bay ◽  
Deborah A Court
Keyword(s):  
Outer Membrane ◽  
Structural Information ◽  
Mitochondrial Outer Membrane ◽  
Wild Type ◽  
Mitochondrial Porin ◽  
Electrophysiological Properties ◽  
Common Structure ◽  
Outer Membrane Porins ◽  
Voltage Dependent ◽  
Bacterial Porins

Voltage-dependent anion-selective channels (VDAC), also known as mitochondrial porins, are key regulators of metabolite flow across the mitochondrial outer membrane. Porins from a wide variety of organisms share remarkably similar electrophysiological properties, in spite of considerable sequence dissimilarity, indicating that they share a common structure. Based on primary sequence considerations, analogy with bacterial porins, and circular dichroism analysis, it is agreed that VDAC spans the outer membrane as a β-barrel. However, the residues that form the antiparallel β-strands comprising this barrel remain unknown. Various predictive methods, largely based on the known structures of bacterial β-barrels, have been applied to the primary sequences of VDAC. Refinement and confirmation of these predictions have developed through numerous investigations of wild-type and variant porins, both in mitochondria and in artificial membranes. These experiments have involved VDAC from several sources, precluding the generation of a unified model. Herein, using the Neurospora VDAC sequence as a template, the published structural information and predictions have been reassessed to delineate a model that satisfies most of the available data.Key words: VDAC, mitochondrial porin, β-barrel.

scholarly journals A molecular link between activation and inactivation of sodium channels.

1995 ◽  
Vol 106 (4) ◽  
pp. 641-658 ◽  
Author(s):  
M E O'Leary ◽  
L Q Chen ◽  
R G Kallen ◽  
R Horn
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Single Channel ◽  
Critical Role ◽  
Channel Measurements ◽  
Wild Type ◽  
Open Probability ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Normal Coupling

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

Identification of the target of gabapentinoid action in neuropathic pain

2018 ◽  
Author(s):  
Turo J. Nurmikko
Keyword(s):  
Neuropathic Pain ◽  
Scientific Community ◽  
Oral Absorption ◽  
Molecular Target ◽  
Wild Type ◽  
Seminal Paper ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels

The landmark paper discussed in this chapter is ‘Identification of the α‎2-δ‎-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin’, published by Field et al. in 2006. In this seminal paper, Field et al. demonstrated that the anti-allodynic effect of pregabalin is related to its binding to the α‎2δ‎-1 subunit of the voltage-gated calcium channel. In transgenic mice lacking this subunit, pregabalin had no effect on allodynia induced by sciatic nerve ligation, whereas, in wild-type mice, there was a substantial anti-allodynic response. This discovery was well received by the scientific community and was considered to conclusively establish the mechanism of action of pregabalin, which has remarkably similar properties to gabapentin but with increased potency and oral absorption. This exciting result acted as an impetus for further studies on the role of the subunit in the development and maintenance of neuropathic pain.

scholarly journals A fern WUSCHEL-RELATED HOMEOBOX gene functions in both gametophyte and sporophyte generations

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Christopher E. Youngstrom ◽  
Lander F. Geadelmann ◽  
Erin E. Irish ◽  
Chi-Lien Cheng
Keyword(s):  
Stem Cells ◽  
Lateral Roots ◽  
Homeobox Gene ◽  
Genetic Networks ◽  
Land Plants ◽  
Seed Plants ◽  
Wild Type ◽  
Cell Fates ◽  
Cell Divisions ◽  
Gene Functions

Abstract Background Post-embryonic growth of land plants originates from meristems. Genetic networks in meristems maintain the stem cells and direct acquisition of cell fates. WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors involved in meristem networks have only been functionally characterized in two evolutionarily distant taxa, mosses and seed plants. This report characterizes a WOX gene in a fern, which is located phylogenetically between the two taxa. Results CrWOXB transcripts were detected in proliferating tissues, including gametophyte and sporophyte meristems of Ceratopteris richardii. In addition, CrWOXB is expressed in archegonia but not the antheridia of gametophytes. Suppression of CrWOXB expression in wild-type RN3 plants by RNAi produced abnormal morphologies of gametophytes and sporophytes. The gametophytes of RNAi lines produced fewer cells, and fewer female gametes compared to wild-type. In the sporophyte generation, RNAi lines produced fewer leaves, pinnae, roots and lateral roots compared to wild-type sporophytes. Conclusions Our results suggest that CrWOXB functions to promote cell divisions and organ development in the gametophyte and sporophyte generations, respectively. CrWOXB is the first intermediate-clade WOX gene shown to function in both generations in land plants.

scholarly journals Regulators of male and female sexual development critical for transmission of a malaria parasite

2021 ◽  
Author(s):  
Andrew J. C. Russell ◽  
Theo Sanderson ◽  
Ellen Bushell ◽  
Arthur M. Talman ◽  
Burcu Anar ◽  
...  
Keyword(s):  
Plasmodium Berghei ◽  
Molecular Mechanisms ◽  
Mosquito Vector ◽  
Wild Type ◽  
Putative Gene ◽  
Regulatory Cascade ◽  
Gene Functions ◽  
Female Sexual Development ◽  
Resolution Single ◽  
Developmental Switch

The transmission of malaria parasites from vertebrate host to mosquito vector requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify ten genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of wild-type and mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain points towards unexpected conservation of molecular mechanisms of gametogenesis in animals and a distantly related eukaryotic parasite.

scholarly journals Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K+ channel

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Anirban Banerjee ◽  
Alice Lee ◽  
Ernest Campbell ◽  
Roderick MacKinnon
Keyword(s):  
Electron Density ◽  
Selectivity Filter ◽  
K Channel ◽  
Wild Type ◽  
Kv Channel ◽  
Pore Blocking ◽  
Kv Channels ◽  
Filter Structure ◽  
Conduction Pathway ◽  
Voltage Dependent

Pore-blocking toxins inhibit voltage-dependent K+ channels (Kv channels) by plugging the ion-conduction pathway. We have solved the crystal structure of paddle chimera, a Kv channel in complex with charybdotoxin (CTX), a pore-blocking toxin. The toxin binds to the extracellular pore entryway without producing discernable alteration of the selectivity filter structure and is oriented to project its Lys27 into the pore. The most extracellular K+ binding site (S1) is devoid of K+ electron-density when wild-type CTX is bound, but K+ density is present to some extent in a Lys27Met mutant. In crystals with Cs+ replacing K+, S1 electron-density is present even in the presence of Lys27, a finding compatible with the differential effects of Cs+ vs K+ on CTX affinity for the channel. Together, these results show that CTX binds to a K+ channel in a lock and key manner and interacts directly with conducting ions inside the selectivity filter.

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