Ca2+ signalling, voltage-gated Ca2+ channels and praziquantel in flatworm neuromusculature.

Transient changes in calcium (Ca2+) levels regulate a wide variety of cellular processes, and cells employ both intracellular and extracellular sources of Ca2+ for signalling. Praziquantel, the drug of choice against schistosomiasis, disrupts Ca2+ homeostasis in adult worms. This review will focus on voltage-gated Ca2+ channels, which regulate levels of intracellular Ca2+ by coupling membrane depolarization to entry of extracellular Ca2+. Ca2+ channels are members of the ion channel superfamily and represent essential components of neurons, muscles and other excitable cells. Ca2+ channels are membrane protein complexes in which the pore-forming alpha1 subunit is modulated by auxiliary subunits such as beta and alpha2delta. Schistosomes express two Ca2+ channel beta subunit subtypes: a conventional subtype similar to beta subunits found in other vertebrates and invertebrates and a novel variant subtype with unusual structural and functional properties. The variant schistosome beta subunit confers praziquantel sensitivity to an otherwise praziquantel-insensitive mammalian Ca2+ channel, implicating it as a mediator of praziquantel action.

Specific sites in the Beta Interaction Domain of a schistosome Ca2+ channel beta subunit are key to its role in sensitivity to the anti-schistosomal drug praziquantel.

Praziquantel, the drug of choice against schistosomiasis, disrupts calcium (Ca2+) homeostasis in schistosomes via an unknown mechanism. Voltage-gated Ca2+ channels are heteromultimeric transmembrane protein complexes that contribute to impulse propagation and also regulate intracellular Ca2+ levels. Beta subunits modulate the properties of the pore-forming alpha1 subunit of high voltage-activated Ca2+ channels. Unlike other Ca2+ channel beta subunits, which have current stimulatory effects, a beta subunit subtype found in S. mansoni (SmbetaA) and S. japonicum (Sjbeta) dramatically reduces current levels when co-expressed with Ca2+ channel alpha1 subunits in Xenopus oocytes. It also confers praziquantel sensitivity to the mammalian Cav2.3 alpha1 subunit. The Beta Interaction Domains (BIDs) of SmbetaA and Sjbeta lack 2 conserved serines that each constitute a consensus site for protein kinase C (PKC) phosphorylation. Here, we use site-directed mutagenesis of schistosome beta subunits to show that these unique functional properties are correlated with the absence of these consensus PKC sites in the BID. Furthermore, a second schistosome beta subunit subtype contains both serines in the BID, enhances currents through alpha1 subunits, and does not confer praziquantel sensitivity. Thus, phosphorylation sites in the BID may play important roles in defining the modulatory properties and pharmacological sensitivities of schistosome Ca2+ channel beta subunits.

Structure of three high voltage-activated calcium channel alpha1 subunits from Schistosoma mansoni.

Voltage-gated calcium (Ca2+) channels contribute to impulse propagation in excitable cells and also regulate intracellular levels of Ca2+. High voltage-activated (HVA) Ca2+ channels are heteromultimeric membrane proteins. The pore-forming, voltage-sensing subunit is the alpha1 subunit. We have cloned 3 HVA Ca2+ channel alpha1 subunit cDNAs from Schistosoma mansoni. One of these sequences most closely resembles the L-type class of HVA alpha1 subunits. The other two sequences are most closely related to non L-type alpha1 subunits. These schistosome alpha1 subunits have many of the features common to HVA Ca2+ channels, but also have distinct structural motifs. Analysis of the structural and functional properties of schistosome Ca2+ channel subunits may provide information about these critical components of excitable cells.

Schistosome calcium channel beta subunits. Unusual modulatory effects and potential role in the action of the antischistosomal drug praziquantel.

Schistosomes are parasitic flatworms that cause schistosomiasis, a major tropical disease. The current drug of choice against schistosomiasis is praziquantel (PZQ), which has minimal side effects and is potent against all schistosome species. The mode of action of PZQ is unknown, though the drug clearly affects Ca(2+) homeostasis in worms, and there is indirect evidence for interaction of PZQ with schistosome voltage-gated Ca(2+) channels. We have cloned and expressed two Ca(2+) channel beta subunits, one from Schistosoma mansoni and one from Schistosoma japonicum. These two subunits (SmCa(v)beta A and SjCa(v)beta) have structural motifs that differ from those found in other known beta subunits. Surprisingly, coexpression of either SmCa(v)beta A or SjCa(v)beta with a cnidarian (CyCa(v)1) or mammalian (Ca(v)2.3) Ca(2+) channel alpha(1) subunit results in a striking reduction in current amplitude. In the case of Ca(v)2.3, this current reduction can be partially reversed by addition of 100 nm PZQ, which results in a significant increase in current amplitude. Thus, these unusual schistosome beta subunits can confer PZQ sensitivity to an otherwise PZQ-insensitive mammalian Ca(2+) channel, indicating that a possible target for PZQ action is the interaction between beta subunits and pore-forming alpha(1) subunits in schistosomes.

Phylogeny of ion channels: clues to structure and function.

Voltage-gated ion channels are responsible for the electrical activity in a variety of cell types in modern-day animals. However, they represent the result of many millions of years of evolution of a family of ion channel proteins that are also found in prokaryotes and diverse eukaryotes, and probably exist in all life forms. This review traces the evolution of ion channels, with particular emphasis on the factors and evolutionary pathways that may have given rise to voltage-gated potassium (K+), calcium (Ca2+), and sodium (Na+) channels. The review also highlights the utility of comparing phylogenetically distinct versions of the same protein as a means to better understand the structure and function of proteins.

Continuation pharmacotherapy in the prevention of relapse following electroconvulsive therapy: a randomized controlled trial.

CONTEXT: Electroconvulsive therapy (ECT) is highly effective for treatment of major depression, but naturalistic studies show a high rate of relapse after discontinuation of ECT. OBJECTIVE: To determine the efficacy of continuation pharmacotherapy with nortriptyline hydrochloride or combination nortriptyline and lithium carbonate in preventing post-ECT relapse. DESIGN: Randomized, double-blind, placebo-controlled trial conducted from 1993 to 1998, stratified by medication resistance or presence of psychotic depression in the index episode. SETTING: Two university-based hospitals and 1 private psychiatric hospital. PATIENTS: Of 290 patients with unipolar major depression recruited through clinical referral who completed an open ECT treatment phase, 159 patients met remitter criteria; 84 remitting patients were eligible and agreed to participate in the continuation study. INTERVENTIONS: Patients were randomly assigned to receive continuation treatment for 24 weeks with placebo (n = 29), nortriptyline (target steady-state level, 75-125 ng/mL) (n = 27), or combination nortriptyline and lithium (target steady-state level, 0.5-0.9 mEq/L) (n = 28). MAIN OUTCOME MEASURE: Relapse of major depressive episode, compared among the 3 continuation groups. RESULTS: Nortriptyline-lithium combination therapy had a marked advantage in time to relapse, superior to both placebo and nortriptyline alone. Over the 24-week trial, the relapse rate for placebo was 84% (95% confidence interval [CI], 70%-99%); for nortriptyline, 60% (95% CI, 41%-79%); and for nortriptyline-lithium, 39% (95% CI, 19%-59%). All but 1 instance of relapse with nortriptyline-lithium occurred within 5 weeks of ECT termination, while relapse continued throughout treatment with placebo or nortriptyline alone. Medication-resistant patients, female patients, and those with more severe depressive symptoms following ECT had more rapid relapse. CONCLUSIONS: Our study indicates that without active treatment, virtually all remitted patients relapse within 6 months of stopping ECT. Monotherapy with nortriptyline has limited efficacy. The combination of nortriptyline and lithium is more effective, but the relapse rate is still high, particularly during the first month of continuation therapy.

Distribution of nitric oxide synthase immunoreactivity in the nervous system and peripheral tissues of Schistosoma mansoni.

The distribution of nitric oxide synthase (NOS) immunoreactivity and putative NOS activity in adult Schistosoma mansoni was analysed using 3 different types of NOS antibodies and NADPH-diaphorase histochemistry. Although potential involvement of the gaseous radical nitric oxide (NO) in host response to infection by schistosomes has been suggested, there is little or no information available regarding the role, or even the presence, of the NO pathway in schistosomes themselves. Here, we demonstrate that antibodies against neuronal NOS (nNOS) and inducible NOS (iNOS) isoforms stain adult worms with distinctive patterns; anti-endothelial NOS (eNOS) shows no selective labelling. nNOS-like immunoreactivity is found in the main nerve cords and the peripheral nervous system. Putative sensory neurons with apical neuronal processes leading to the tegument of male worms are also immunoreactive for nNOS. Anti-iNOS labels a variety of predominantly non-neuronal tissues, showing intense labelling at or near the surface of the worm and in components of the gastrointestinal tract. The distribution of NADPH-diaphorase reactivity (a histochemical marker of NOS), is generally similar to the pattern of NOS immunoreactivity, including labelling of neuronal-like cells as well as developing eggs. These results suggest that an NOS-like enzyme is present in S. mansoni, and indicate potential roles for the different NOS isoforms in neuronal signalling, reproduction and development.

Calcium channel beta subunits differentially modulate recovery of the channel from inactivation.

We examined the effects of calcium channel beta subunits upon the recovery from inactivation of alpha(1) subunits expressed in Xenopus oocytes. Recovery of the current carried by the L-type alpha(1) subunit (cyCa(v)1) from the jellyfish Cyanea capillata was accelerated by coexpression of any beta subunit, but the degree of potentiation differed according to which beta isoform was coexpressed. The Cyanea beta subunit was most effective, followed by the mammalian b(3), b(4), and beta(2a) subtypes. Recovery of the human Ca(v)2.3 subunit was also modulated by beta subunits, but was slowed instead. beta(3) was the most potent subunit tested, followed by beta(4), then beta(2a), which had virtually no effect. These results demonstrate that different beta subunit isoforms can affect recovery of the channel to varying degrees, and provide an additional mechanism by which beta subunits can differentially regulate alpha(1) subunits.

Characterization of a phosphoinositide-mediated odor transduction pathway reveals plasma membrane localization of an inositol 1,4, 5-trisphosphate receptor in lobster olfactory receptor neurons.

The role of phosphoinositide signaling in olfactory transduction is still being resolved. Compelling functional evidence for the transduction of odor signals via phosphoinositide pathways in olfactory transduction comes from invertebrate olfactory systems, in particular lobster olfactory receptor neurons. We now provide molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons, a G protein alpha subunit of the G(q) family and an inositol 1,4, 5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP(3)) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP(3) receptor localizes to membranes in the ciliary transduction compartment of these cells at both the light microscopic and electron microscopic levels. Given the absence of intracellular organelles in the sub-micron diameter olfactory cilia, this finding indicates that the IP(3) receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP(3)R in neurons. The association of the IP(3) receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.

Determinants of seizure threshold in ECT: benzodiazepine use, anesthetic dosage, and other factors.

The electrical dosage of the ECT stimulus impacts on efficacy and cognitive side effects, yet seizure threshold (ST) may vary as much as 50-fold across patients. It would be desirable to predict ST on the basis of patient and treatment characteristics. In particular, concerns have been raised that benzodiazepine use and higher dosage of barbiturate anesthetics elevate ST. In a three-site study, ST was quantified at the first ECT session using an identical empirical titration procedure in 294 patients who met RDC and DSM-IIIR criteria for a major depressive episode. ST varied over a 35-fold range across patients treated with right unilateral (RUL) (n = 267) and bilateral (BL) (n = 27) ECT. Higher ST was associated with BL electrode placement (p = 0.001). Among patients treated with RUL ECT, univariate analyses indicated that higher ST was associated with advanced age (p < 0.001), male gender (p < 0.001), greater burden of medical illness (p < 0.001), weight (p < 0.01), duration of mood disorder (p < 0.01), and history of previous ECT (p < 0.05). Average lorazepam dose in the 48 hours prior to ECT was not associated with ST, but was associated with decreased seizure duration (p < 0.01). Absolute, but not weight-adjusted, methohexital dose was associated with ST (p < 0.01). Multivariate analyses in patients treated with unilateral ECT showed that only 27.6% of the variance in ST (p < 0.0001) could be predicted. In the multivariate analyses, only age (p = 0.0001), gender (p = 0.01), and methohexital dose (p = 0.0001) were independently related to ST. Low dosage of lorazepam and methohexital dosage below 1 mg/kg are unlikely to impact on ST. Given the limited capacity to predict ST, empirical titration remains the only accurate method to determine electrical dosage in RUL ECT.

The molecular biology of invertebrate voltage-gated Ca(2+) channels.

The importance of voltage-gated Ca(2+) channels in cellular function is illustrated by the many distinct types of Ca(2+) currents found in vertebrate tissues, a variety that is generated in part by numerous genes encoding Ca(2+) channel subunits. The degree to which this genetic diversity is shared by invertebrates has only recently become apparent. Cloning of Ca(2+) channel subunits from various invertebrate species, combined with the wealth of information from the Caenorhabditis elegans genome, has clarified the organization and evolution of metazoan Ca(2+) channel genes. Functional studies have employed novel structural information gained from invertebrate Ca(2+) channels to complement ongoing research on mammalian Ca(2+) currents, while demonstrating that the strict correspondence between pharmacological and molecular classes of vertebrate Ca(2+) channels does not fully extend to invertebrate tissues. Molecular structures can now be combined with physiological data to develop a more cogent system of categorizing invertebrate channel subtypes. In this review, we examine recent progress in the characterization of invertebrate Ca(2+) channel genes and its relevance to the diversity of invertebrate Ca(2+) currents.

Cloning and expression of a jellyfish calcium channel beta subunit reveal functional conservation of the alpha1-beta interaction.

In high voltage-activated calcium channels, the binding between the pore-forming alpha1 subunit and the modulatory beta subunit is mediated by interaction domains in each molecule that are highly conserved among most known subunits. However, the interaction domain within CyCaalpha1, an alpha1 subunit cloned from the jellyfish Cyanea capillata, matches the canonical sequence of the alpha1 interaction domain at only four of nine sites. We have now cloned a cDNA from Cyanea neuromuscular tissue that encodes a Ca2+ channel beta subunit. The subunit, named CyCabeta, shares 47-54% identity with vertebrate beta subunit isoforms, but is most highly conserved within its interaction domain. Coexpression of CyCabeta with CyCaalpha1 in Xenopus oocytes increases the amplitude of the CyCaalpha1 current and shifts its activation to more hyperpolarized potentials. These responses are mimicked by coexpression of the rat beta2a subunit, demonstrating that the alpha1 beta interaction is functionally conserved between cnidarians and mammals. CyCabeta also markedly accelerates the rate of recovery of CyCaalpha1 from inactivation, an action that is modestly duplicated by beta2a and may represent an additional mechanism by which beta subunit isoforms differentially modulate alpha1 subunits. These findings establish that limited conservation within the alpha1 interaction domain is sufficient to allow full modulation by a beta subunit, as well as altered regulation by different beta isoforms.

Diagnostic value of a pacemaker high rate episode counter when programming the ventricular tachycardia zone of an ICD.

A 72-year-old man with an ICD and a pacemaker was presented with an episode of sustained VT that accelerated to VF. The ICD failed to detect the event and deliver therapy, despite a VT apparently within the VT detection zone. The ICD detected the event after degeneration to VF and delivered appropriate therapy. The high rate event feature of the pacemaker was useful in determining proper function of the ICD along with optimal programming of VT detection.

Use of atrial and ventricular electrograms from a dual chamber implantable cardioverter defibrillator to elucidate a complex dysrhythmia.

Stored intracardiac electrograms provided by third-generation ICDs have proved their use in the analysis of the mechanism of tachydysrhythmic events. There are cases in which the analysis of ventricular electrograms is insufficient for the elucidation of certain dysrhythmias. The availability of atrial electrograms provided by dual chamber ICDs improves the diagnostic capability of electrogram analysis and could prove most useful especially in complex dysrhythmias.

Cloning and functional expression of a voltage-gated calcium channel alpha1 subunit from jellyfish.

Voltage-gated Ca2+ channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel alpha1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the alpha1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfish Cyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaalpha1, is more similar to vertebrate L-type channels (alpha1S, alpha1C, and alpha1D) than to non-L-type channels (alpha1A, alpha1B, and alpha1E) or low voltage-activated channels (alpha1G). Expression of CyCaalpha1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrate L-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(-)-BayK 8644. In addition, the channel is less permeable to Ba2+ than to Ca2+ and is more permeable to Sr2+. CyCaalpha1 thus represents an ancestral L-type alpha1 subunit with significant functional differences from mammalian L-type channels.

A myosin III from Limulus eyes is a clock-regulated phosphoprotein.

The lateral eyes of the horseshoe crab Limulus polyphemus undergo dramatic daily changes in structure and function that lead to enhanced retinal sensitivity and responsiveness to light at night. These changes are controlled by a circadian neural input that alters photoreceptor and pigment cell shape, pigment migration, and phototransduction. Clock input to the eyes also regulates photomechanical movements within photoreceptors, including membrane shedding. The biochemical mechanisms underlying these diverse effects of the clock on the retina are unknown, but a major biochemical consequence of activating clock input to the eyes is a rise in the concentration of cAMP in photoreceptors and the phosphorylation of a 122 kDa visual system-specific protein. We have cloned and sequenced cDNA encoding the clock-regulated 122 kDa phosphoprotein and show here that it is a new member of the myosin III family. We report that Limulus myosin III is similar to other unconventional myosins in that it binds to calmodulin in the absence of Ca2+; it is novel in that it is phosphorylated within its myosin globular head, probably by cAMP-dependent protein kinase. The protein is present throughout the photoreceptor, including the region occupied by the photosensitive rhabdom. We propose that the phosphorylation of Limulus myosin III is involved in one or more of the structural and functional changes that occur in Limulus eyes in response to clock input.

Structure of a putative sodium channel from the sea anemone Aiptasia pallida.

A cDNA encoding a full length putative sodium channel has been cloned from the sea anemone Aiptasia pallida. The deduced protein, named AiNa1, has a predicted molecular weight of 205,000 Da. It shows high structural similarity to other sodium channels from both invertebrates and vertebrates, and its structure is consistent with the four domain, six transmembrane segment motif of all known voltage-gated sodium channels. In the region purported to constitute the tetrodotoxin (TTX) receptor of sodium channels, AiNa1 differs from the TTX-sensitive motif, suggesting that currents carried by this channel would be insensitive to TTX. The presence of a conventional sodium channel protein in anemones indicates, for the first time, that neurons in sea anemones are likely to be capable of producing fast, overshooting action potentials.

Cloning of a putative voltage-gated sodium channel from the turbellarian flatworm Bdelloura candida.

The neuromuscular sodium currents of early invertebrates such as platyhelminths display distinctive kinetic and pharmacological properties. We have cloned a cDNA from the horseshoe crab flatworm Bdelloura candida that encodes a protein homologous to the primary subunit of voltage-gated sodium channels. The B. candida protein, named BdNa1, exhibits amino acid identity of 40-47% to sodium channels of vertebrates and higher invertebrates. BdNa1 has the multidomain structure characteristic of sodium channels, and is most highly conserved in the hydrophobic transmembrane segments and the regions that form the pore of the channel. Northern blot analysis confirms the presence of a 5.4 kb BdNa1 transcript in B. candida tissue. The information provided by analysis of the BdNa1 sequence offers insight into the physiology of platyhelminth sodium currents.