The AKT2 potassium channel mediates NaCl induced depolarization in the root of Arabidopsis thaliana.

Soil salinization is a major cause of plant stress, partly due to the physicochemical similarities between Na(+) and K(+). Na(+) ions compete with K(+) ions for their transport into root cells. However, the point of Na(+) entry remains unidentified. Here, I have applied the Electrical Penetration Graph as a method for whole plant electrophysiology in order to test if (a) root exposure to NaCl induces depolarization waves that propagate from root to shoot via the phloem, and if (b) the electrophysiological effects of root exposure to NaCl require expression of the potassium channels AKT1 and/or AKT2. The data suggest that AKT2 subunit containing K(+) channels mediate NaCl-induced depolarization of root cells, and that this depolarization does not propagate to leaves via the phloem.

New roles for the GLUTAMATE RECEPTOR-LIKE 3.3, 3.5, and 3.6 genes as on/off switches of wound-induced systemic electrical signals.

Wounding induces systemic potentials in Arabidopsis thaliana that can be abolished by concomitant suppression of the GLUTAMATE RECEPTOR-LIKE GLR3.3 and GLR3.6 genes. However, the roles of specific GLR channels to these potentials remain unclear. Here I applied the Electrical Penetration Graph (EPG) to study the contribution of three GLR channels to wound-induced, systemically propagated electrical potentials in Arabidopsis thaliana. In contrast to recordings made with conventional rigs for whole-plant electrophysiology, the EPG allows for the unambiguous distinction of the phloem-propagated action potential (AP) from the electrical activity outside of the phloem. The data reported here suggest that: (a) the transmission of wound-induced, phloem-propagated AP to neighbor leaves, requires expression of GLR3.3 or GLR3.6, whereas GLR3.5 prevents its transmission to non-neighbor leaves; (b) the generation of wound-induced electrical signals outside the phloem network depends on GLR3.6 expression; and (c) wound-induced systemic potentials initiated in the shoot are transmitted to the root in the adult plant, which suggests a role for these electrical signals in coordinating the plant defenses in the shoot and in the root. Here, I propose a model for wound-induced systemic electrical signals at the molecular, cellular and anatomical level. In this model, GLR3.3 and GLR3.6 function as on switches for the propagation of wound-induced potentials beyond the wounded leaf, while GLR3.5 functions as an off switch that prevents the propagation of wound-induced electrical potentials to distal, non-neighbor leaves.

Electrical Wiring and Long-Distance Plant Communication.

Electrical signalling over long distances is an efficient way of achieving cell-to-cell communication in living organisms. In plants, the phloem can be considered as a 'green cable' that allows the transmission of action potentials (APs) induced by stimuli such as wounding and cold. Measuring phloem potential changes and separating them from secondary responses of surrounding tissues can be achieved using living aphids as bioelectrodes. Two glutamate receptor-like genes (GLR3.3 and 3.6) were identified as being involved in the propagation of electrical activity from the damaged to undamaged leaves. However, phloem APs are initiated and propagated independently of these glutamate receptors. Here, we propose new screening approaches to obtain further information on the components required for electrical signalling in phloem cables.

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements.

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana(1). Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

Real-time, in vivo intracellular recordings of caterpillar-induced depolarization waves in sieve elements using aphid electrodes.

Plants propagate electrical signals in response to artificial wounding. However, little is known about the electrophysiological responses of the phloem to wounding, and whether natural damaging stimuli induce propagating electrical signals in this tissue. Here, we used living aphids and the direct current (DC) version of the electrical penetration graph (EPG) to detect changes in the membrane potential of Arabidopsis sieve elements (SEs) during caterpillar wounding. Feeding wounds in the lamina induced fast depolarization waves in the affected leaf, rising to maximum amplitude (c. 60 mV) within 2 s. Major damage to the midvein induced fast and slow depolarization waves in unwounded neighbor leaves, but only slow depolarization waves in non-neighbor leaves. The slow depolarization waves rose to maximum amplitude (c. 30 mV) within 14 s. Expression of a jasmonate-responsive gene was detected in leaves in which SEs displayed fast depolarization waves. No electrical signals were detected in SEs of unwounded neighbor leaves of plants with suppressed expression of GLR3.3 and GLR3.6. EPG applied as a novel approach to plant electrophysiology allows cell-specific, robust, real-time monitoring of early electrophysiological responses in plant cells to damage, and is potentially applicable to a broad range of plant-herbivore interactions.

Calcium channels of schistosomes: unresolved questions and unexpected answers.

Parasitic flatworms of the genus Schistosoma are the causative agents of schistosomiasis, a highly prevalent, neglected tropical disease that causes significant morbidity in hundreds of millions of people worldwide. The current treatment of choice against schistosomiasis is praziquantel (PZQ), which is known to affect Ca(2+) homeostasis in schistosomes, but which has an undefined molecular target and mode of action. PZQ is the only available antischistosomal drug in most parts of the world, making reports of PZQ resistance particularly troubling. Voltage-gated Ca(2+) (Ca(v)) channels have been proposed as possible targets for PZQ, and, given their central role in the neuromuscular system, may also serve as targets for new anthelmintic therapeutics. Indeed, ion channels constitute the majority of targets for current anthelmintics. Ca(v) channel subunits from schistosomes and other platyhelminths have several unique properties that make them attractive as potential drug targets, and that could also provide insights into structure-function relationships in, and evolution of, Ca(v) channels.

The N terminus of a schistosome beta subunit regulates inactivation and current density of a Cav2 channel.

The ß subunit of high voltage-activated Ca(2+) (Ca(v)) channels targets the pore-forming α(1) subunit to the plasma membrane and tunes the biophysical phenotype of the Ca(v) channel complex. We used a combination of molecular biology and whole-cell patch clamp to investigate the functional role of a long N-terminal polyacidic motif (NPAM) in a Ca(v)ß subunit of the human parasite Schistosoma mansoni (ß(Sm)), a motif that does not occur in other known Ca(v)ß subunits. When expressed in human embryonic kidney cells stably expressing Ca(v)2.3, ß(Sm) accelerates Ca(2+)/calmodulin-independent inactivation of Ca(v)2.3. Deleting the first 44 amino acids of ß(Sm), a region that includes NPAM, significantly slows the predominant time constant of inactivation (τ(fast)) under conditions that prevent Ca(2+)/CaM-dependent inactivation (ß(Sm): τ(fast) = 66 ms; ß(SmΔ2-44): τ(fast) = 111 ms, p < 0.01). Interestingly, deleting the amino acids that are N-terminal to NPAM (2-24 or 2-17) results in faster inactivation than with an intact N terminus (τ(fast) = 42 ms with ß(SmΔ2-17); τ(fast) = 40 ms with ß(SmΔ2-24), p < 0.01). This suggests that NPAM is the structural determinant for accelerating Ca(2+)/calmodulin-independent inactivation. We also created three chimeric subunits that contain the first 44 amino acids of ß(Sm) attached to mammalian ß(1b), ß(2a), and ß(3) subunits. For any given mammalian ß subunit, inactivation was faster if it contained the N terminus of ß(Sm) than if it did not. Co-expression of the mammalian α(2)δ-1 subunit resulted in doubling of the inactivation rate, but the effects of NPAM persisted. Thus, it appears that the schistosome Ca(v) channel complex has acquired a new function that likely contributes to reducing the amount of Ca(2+) that enters the cells in vivo. This feature is of potential interest as a target for new antihelminthics.

Specific and slow inhibition of the kir2.1 K+ channel by gambogic acid.

Although Kir2.1 channels are important in the heart and other excitable cells, there are virtually no specific drugs for this K+ channel. In search of Kir2.1 modulators, we screened a library of 720 naturally occurring compounds using a yeast strain in which mammalian Kir2.1 enables growth at low [K+]. One of the identified compounds, gambogic acid (GA), potently (EC(50) < or = 100 nm) inhibited Kir2.1 channels in mammalian cells when applied chronically for 3 h. This potent and slow inhibition was not seen with Kv2.1, HERG or Kir1.1 channels. However, acutely applied GA acted as a weak (EC(50) = approximately 10 mum) non-selective K+ channel blocker. Intracellular delivery of GA via a patch pipette did not potentiate the acute effect of GA on Kir2.1, showing that slow uptake is not responsible for the delayed, potent effect. Immunoblots showed that total Kir2.1 protein expression was not altered by GA. Similarly, immunostaining of intact cells expressing Kir2.1 with an extracellular epitope tag demonstrated that GA does not affect Kir2.1 surface expression. However, the 3-h treatment with GA caused redistribution of Kir2.1 and Kv2.1 from the Triton X-100-insoluble to the Triton X-100-soluble membrane fraction. Thus, GA changes the K+ channel membrane microenvironment resulting in potent, specific, and slow acting inhibition of Kir2.1 channels.

Atypical properties of a conventional calcium channel beta subunit from the platyhelminth Schistosoma mansoni.

BACKGROUND: The function of voltage-gated calcium (Cav) channels greatly depends on coupling to cytoplasmic accessory beta subunits, which not only promote surface expression, but also modulate gating and kinetic properties of the alpha1 subunit. Schistosomes, parasitic platyhelminths that cause schistosomiasis, express two beta subunit subtypes: a structurally conventional beta subunit and a variant beta subunit with unusual functional properties. We have previously characterized the functional properties of the variant Cavbeta subunit. Here, we focus on the modulatory phenotype of the conventional Cavbeta subunit (SmCavbeta) using the human Cav2.3 channel as the substrate for SmCavbeta and the whole-cell patch-clamp technique. RESULTS: The conventional Schistosoma mansoni Cavbeta subunit markedly increases Cav2.3 currents, slows macroscopic inactivation and shifts steady state inactivation in the hyperpolarizing direction. However, currents produced by Cav2.3 in the presence of SmCavbeta run-down to approximately 75% of their initial amplitudes within two minutes of establishing the whole-cell configuration. This suppressive effect was independent of Ca2+, but dependent on intracellular Mg2+-ATP. Additional experiments revealed that SmCavbeta lends the Cav2.3/SmCavbeta complex sensitivity to Na+ ions. A mutant version of the Cavbeta subunit lacking the first forty-six amino acids, including a string of twenty-two acidic residues, no longer conferred sensitivity to intracellular Mg2+-ATP and Na+ ions, while continuing to show wild type modulation of current amplitude and inactivation of Cav2.3. CONCLUSION: The data presented in this article provide insights into novel mechanisms employed by platyhelminth Cavbeta subunits to modulate voltage-gated Ca2+ currents that indicate interactions between the Ca2+ channel complex and chelated forms of ATP as well as Na+ ions. These results have potentially important implications for understanding previously unknown mechanisms by which platyhelminths and perhaps other organisms modulate Ca2+ currents in excitable cells.

Voltage-gated k+ channel block by catechol derivatives: defining nonselective and selective pharmacophores.

High-throughput screening led to the identification of a 3-norbornyl derivative of catechol called 48F10 (3-bicyclo[2.2.1]hept-2-yl-benzene-1,2-diol) as a Kv2.1 K(+) channel inhibitor. By virtue of the involvement of Kv2.1 channels in programmed cell death, 48F10 prevents apoptosis in cortical neurons and enterocytes. This uncharged compound acts with an apparent affinity of 1 muM at the tetraethylammonium (TEA) site at the external mouth of the Kv2.1 channel but is ineffective on Kv1.5. Here we investigated the basis of this selectivity with structure-activity studies. We find that catechol (1,2-benzenediol), unlike 48F10, inhibits Kv2.1 currents with a Hill coefficient of 2 and slows channel activation. Furthermore, this inhibition, which requires millimolar concentrations, is unaffected by external TEA or by mutation of the external tyrosine implicated in channel block by TEA and 48F10. In addition, catechol does not distinguish between Kv2.1 and Kv1.5. Thus, catechol acts at conserved sites that are distinct from 48F10. We also tested 11 catechol derivatives based on hydrocarbon adducts including norbornyl substructures, a 48F10 isomer, and a 48F10 diastereomer. These compounds are more potent than catechol, but none replicated the marked selectivity of 48F10 for Kv2.1 over Kv1.5. We conclude that the targeting of 48F10 to the TEA site at the external mouth of the Kv2.1 pore and away from other sites involved in nonselective Kv channel block by catechol requires the norbornyl group in a unique position and orientation on the catechol ring.

A potassium channel (Kv4) cloned from the heart of the tunicate Ciona intestinalis and its modulation by a KChIP subunit.

Voltage-gated ion channels of the Kv4 subfamily produce A-type currents whose properties are tuned by accessory subunits termed KChIPs, which are a family of Ca2+ sensor proteins. By modifying expression levels and the intrinsic biophysical properties of Kv4 channels, KChIPs modulate the excitability properties of neurons and myocytes. We studied how a Kv4 channel from a tunicate, the first branching clade of the chordates, is modulated by endogenous KChIP subunits. BLAST searches in the genome of Ciona intestinalis identified a single Kv4 gene and a single KChIP gene, implying that the diversification of both genes occurred during early vertebrate evolution, since the corresponding mammalian gene families are formed by several paralogues. In this study we describe the cloning and characterization of a tunicate Kv4 channel, CionaKv4, and a tunicate KChIP subunit, CionaKChIP. We demonstrate that CionaKChIP strongly modulates CionaKv4 by producing larger currents that inactivate more slowly than in the absence of the KChIP subunit. Furthermore, CionaKChIP shifted the midpoints of activation and inactivation and slowed deactivation and recovery from inactivation of CionaKv4. Modulation by CionaKChIP requires the presence of the intact N terminus of CionaKv4 because, except for a minor effect on inactivation, CionaKChIP did not modulate CionaKv4 channels that lacked amino acids 2-32. In summary, our results suggest that modulation of Kv4 channels by KChIP subunits is an ancient mechanism for modulating electrical excitability.

Attenuation of apoptosis in enterocytes by blockade of potassium channels.

Apoptosis plays an important role in maintaining the balance between proliferation and cell loss in the intestinal epithelium. Apoptosis rates may increase in intestinal pathologies such as inflammatory bowel disease and necrotizing enterocolitis, suggesting pharmacological prevention of apoptosis as a therapy for these conditions. Here, we explore the feasibility of this approach using the rat epithelial cell line IEC-6 as a model. On the basis of the known role of K+ efflux in apoptosis in various cell types, we hypothesized that K+ efflux is essential for apoptosis in enterocytes and that pharmacological blockade of this efflux would inhibit apoptosis. By probing intracellular [K+] with the K+-sensitive fluorescent dye and measuring the efflux of 86Rb+, we found that apoptosis-inducing treatment with the proteasome inhibitor MG-132 leads to a twofold increase in K+ efflux from IEC-6 cells. Blockade of K+ efflux with tetraethylammonium, 4-aminopyridine, stromatoxin, chromanol 293B, and the recently described K+ channel inhibitor 48F10 prevents DNA fragmentation, caspase activation, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential. Thus K+ efflux occurs early in the apoptotic program and is required for the execution of later events. Apoptotic K+ efflux critically depends on activation of p38 MAPK. These results demonstrate for the first time the requirement of K+ channel-mediated K+ efflux for progression of apoptosis in enterocytes and suggest the use of K+ channel blockers to prevent apoptotic cell loss occurring in intestinal pathologies.