Cation-selective Ion Channels Research Articles

Five nicotinic acetylcholine receptor subunits from the Morotoge shrimp, Pandalopsis japonica: cloning, tissue distribution, and functional expression in Xenopus oocytes

The nicotinic acetylcholine receptor (nAChR) is a member of the ligand-gated ion channel (LGIC) family and is composed of five subunits arranged around a central pore. Expressed sequence tag screening and traditional cloning strategies revealed five full-length cDNAs encoding nAChR subunit homologs (Pajα3, Pajα10, Pajα11, Pajα12, and Pajβ1) in the Morotoge shrimp, Pandalopsis japonica. The nAChR subunits exhibited common structural characteristics, including a signal peptide sequence, a large N-terminal extracellular domain with conserved motifs for ligand binding (loops A–F), and a transmembrane (TM) domain with four hydrophobic TM motifs (TM1–TM4). Based on the conserved GEK motifs located just before TM2, all five nAChR subunits from P. japonica appear to be cation-selective ion channels. Among the five subunits, Pajα3 and Pajβ1 clustered together with insect core groups, whereas Pajα10, Pajα11, and Pajα12 were classified as a divergent group. Three distinct transcripts were identified in Pajα3, presumably due to alternative splicing between TM3 and TM4, which may be involved in channel formation with other subunits. All five nAChR subunits were expressed predominantly in neuronal tissues, including the brain, sinus gland/X-organ complex, thoracic ganglia, and abdominal ganglia, with no significant differences in subunit expression levels among the neuronal tissues. The five shrimp nAChR subunits could not be functionally expressed in Xenopus oocytes, but coexpression of Pajβ1 and rat α4 subunit (Rα4) formed functional channels responding to acetylcholine. Functional expression of vertebrate α subunit (Rα4) with invertebrate β1 subunit (Pajβ1) will expand our knowledge regarding the structural characteristics and molecular gating mechanism of invertebrate nAChRs.

Voltage-dependent cation-selective ion channels formed by peroxiredoxin 6 in a lipid bilayer

The formation of clusters with cation-selective ion-channel properties by the antioxidant enzyme peroxiredoxin-6 that is incorporated into a phosphatidylcholine lipid bilayer has been demonstrated experimentally for the first time. The oligomeric clusters of channels contained three or more individual channel subunits with a conductivity of approximately 350 pS in 200 mM KCl. The average “open” time of the channel cluster decreased as the membrane potential increased. The molecular mechanisms of inactivation of the channel clusters are considered.

Targeting TRP channels for chronic cough: from bench to bedside.

Cough is currently the most common reason for patients to visit a primary care physician in the UK, yet it remains an unmet medical need. Current therapies have limited efficacy or have potentially dangerous side effects. Under normal circumstances, cough is a protective reflex to clear the lungs of harmful particles; however, in disease, cough can become excessive, dramatically impacting patients' lives. In many cases, this condition is linked to inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD), but can also be refractory to treatment and idiopathic in nature. Therefore, there is an urgent need to develop therapies, and targeting the sensory afferent arm of the reflex which initiates the cough reflex may uncover novel therapeutic targets. The cough reflex is initiated following activation of ion channels present on vagal sensory afferents. These ion channels include the transient receptor potential (TRP) family of cation-selective ion channels which act as cellular sensors and respond to changes in the external environment. Many direct activators of TRP channels, including arachidonic acid derivatives, a lowered airway pH, changes in temperature, and altered airway osmolarity are present in the diseased airway where responses to challenge agents which activate airway sensory nerve activity are known to be enhanced. Furthermore, the expression of some TRP channels is increased in airway disease. Together, this makes them promising targets for the treatment of chronic cough. This review will cover the current understanding of the role of the TRP family of ion channels in the activation of airway sensory nerves and cough, focusing on four members, transient receptor potential vanilloid (TRPV) 1, transient receptor potential ankyrin (TRPA) 1, TRPV4, and transient receptor potential melastatin (TRPM) 8 as these represent the channels where most information has been gathered with relevance to the airways. We will describe recent data and highlight the possible therapeutic utility of specific TRP channel antagonists as antitussives in the clinic.

Probing the Mechanosensitivity of Piezo1 Channels

Piezo channels are a new family of cation-selective mechanosensitive ion channels, which have been shown to be integral in numerous physiological processes. Central to these physiological roles is the ability to sense mechanical force. Whether this force is transmitted directly from the lipid bilayer or from tethering to the cytoskeleton and/or ECM is unknown. The ‘gold standard’ for testing the ‘inherent’ mechanosensitivity of ion channels has become purification and subsequent reconstitution of mechanosensitive channel proteins into liposomes of known lipid composition. This is experimentally difficult and time consuming and is a process that has to be determined empirically for individual channel proteins. An underused paradigm for the study of channel mechanosensitivity is the production of cytoskeleton deficient membrane ‘blebs’ [1]. In order to study the inherent mechanosensitivity of Piezo1 channels using this paradigm we used transiently transfected HEK293 cells. Herein we show that; a) membrane blebs can be formed by the addition of both Hypo- and Hyper-osmolar solutions of sodium gluconate in HEK293 cells, b) this treatment induces significant cell death after 2 hours as determined by trypan blue exclusion assays, c) these resulting blebs are deficient in a major cytoskeletal component (actin) and d) Piezo1 channels can be activated in this environment and have a lower pressure threshold of activation. This has important implications with respect to Piezo1 channels being gated according to the force from lipids concept [2]. In addition we also assess the affect of mutating a number of aromatic residues in the most highly conserved region of the Piezo sequence on the mechanosensitivity of the channel.

Abstract 12292: GsMTx4-D is a Cardioprotectant Against Myocardial Infarction During Ischemia and Reperfusion

Introduction: Reperfusion of ischemic cardiac tissue by percutaneous coronary intervention has become a routine treatment for acute ST-elevation myocardial infarction. However, restoring blood flow to ischemic cardiac tissue can injure myocytes that were viable prior to reperfusion. The putative causes of ischemia/reperfusion (I/R) injury are varied, though cation imbalance has long been known to be a significant contributor. GsMTx4 peptide is a selective inhibitor of cation selective mechanosensitive ion channels (MSCs) and has helped establish the role of MSCs in cardiac physiology. However, the role of MSCs in ischemic reperfusion injury remain uncertain. Hypothesis: Pathological stresses from swelling and hypercontracture during I/R can activate cationic MSCs, the D enantiomer of GsMTx4 can act as a cardioprotectant during I/R. Methods: GsMTx4-D pharmacokinetics in the plasma and heart was monitored over 24 h using an LCMSMS assay. The cardioprotective activities of GsMTx4 were monitored in a mouse model of ischemia/reperfusion (I/R) injury in which the left anterior descending coronary artery (LAD) was occluded and then released. The influence of GsMTx4 on contractile function and transient calcium signaling of cardiomyocyte were assessed by SoftEdge MyoCam system. Results: GsMTx4-D reduced infarct area by >40% for both acute and pretreated animals relative to vehicle injected animals (p<0.01). Cardiac output was significantly improved in GsMTx4-D pretreatment group, and arrhythmias were also significantly reduced by intravenous injections of GsMTx4-D. The further studies with isolated cardiomyocytes revealed that GsMTx4-D improved contractility and intracellular transient calcium signaling of cardiomyocytes under hypoxia/reoxygenation conditions. The immunoblotting data demonstrated that administration of GsMTx4-D attenuated I/R-induced inflammatory c-Jun N-terminal protein kinase (JNK) and NF-κB signaling pathways in the hearts. Conclusions: GsMTx4-D is a potent cardioprotectant that decreases infarct area, improves heart function, reduces arrhythmias and attenuates inflammatory signaling pathways that are caused by I/R. Thus GsMTx4-D could be a useful therapy in treating cardiac I/R injury.

Abstract 341: GsMTx4-D is a Cardioprotectant against Myocardial Infarction during Ischemia and Reperfusion

Rationale: GsMTx4 peptide is a selective inhibitor of cation selective mechanosensitive ion channels (MSCs) and has helped establish the role of MSCs in cardiac physiology. However, the role of MSCs in ischemic reperfusion injury was unknown. Cation imbalance appears to be a major contributor to ischemic reperfusion injury across multiple systems. MSCs may be significant contributors to the cation overload, and they are likely to be activated in reperfusion due to inhomogeneous local stresses and compromised mechanical support of the sarcolemma. Objective: The aim of this study was to determine if the D enantiomer of GsMTx4 can act as a cardioprotectant during ischemia/reperfusion in mice. Methods and Results: GsMTx4-D pharmacokinetics in the plasma and heart was monitored over 24 hrs using an LCMSMS assay. Ischemia and reperfusion in the mouse heart involved ligating a coronary artery for 20 min followed by release of the ligature. GsMTx4-D was administered by either acute intravenous injection during the ischemic event, or a day prior to the event with two days of intraperitoneal injections, once per day. Dosing of GsMTx4-D was adjusted to achieve tissue concentrations of 1-5 μM. Relative to vehicle injected animals, GsMTx4-D reduced infarct area by >40% for both acute and pretreated animals. Cardiac output was indistinguishable between sham-treated control hearts and GsMTx4-D pretreatment, and arrhythmias were also significantly reduced by intravenous injections of GsMTx4-D. Conclusions: GsMTx4-D is a potent cardioprotectant that decreases infarct area, increases cardiac output and decreases arrhythmias that are caused by ischemia and reperfusion. GsMTx4-D was not toxic and promises to be a useful therapy in reperfusion of the heart and other organs.

Transient receptor potential channels as drug targets: from the science of basic research to the art of medicine.

The large Trp gene family encodes transient receptor potential (TRP) proteins that form novel cation-selective ion channels. In mammals, 28 Trp channel genes have been identified. TRP proteins exhibit diverse permeation and gating properties and are involved in a plethora of physiologic functions with a strong impact on cellular sensing and signaling pathways. Indeed, mutations in human genes encoding TRP channels, the so-called "TRP channelopathies," are responsible for a number of hereditary diseases that affect the musculoskeletal, cardiovascular, genitourinary, and nervous systems. This review gives an overview of the functional properties of mammalian TRP channels, describes their roles in acquired and hereditary diseases, and discusses their potential as drug targets for therapeutic intervention.

TRPM5 is a target in the modulation of taste perception and insulin secretion (133.4)

TRPM5 is a monovalent cation selective ion channels, activated by an increase of the intracellular Ca2+ concentration. Physiologically, TRPM5 is a key player in the transduction of taste signals and plays a role in the release of insulin from pancreatic ß‐cells. To date no pharmacological tools are available to evaluate the potential of TRPM5 as drug target for taste modulation or as insulin secretagogue. Here we show that the TRPM5 ion channel has potential as a target to stimulate insulin secretion in a mouse model We present a new class of compounds capable of potentiating TRPM5 signaling and analyze their working mechanism. We show activation of TRPM5 in a heterologous overexpression system and in a murine animal model. We show potentiation of sweet and bitter taste in a TRPM5 dependent way, using a two bottle preference test. Furthermore we observe increased glucose induced activity in the insulin secreting islets of Langerhans upon perfusion with our activator. These effects were completely abolished in TRPM5 knock‐out mice. In the high fat fed mouse, a model for type II diabetes, we showed that activating TRPM5 with our compound allows them to maintain a healthier glucose metabolism and delays the development of diabetes.

Absence of Transient Receptor Potential Vanilloid-1 Accelerates Stress-Induced Axonopathy in the Optic Projection

How neurons respond to stress in degenerative disease is of fundamental importance for identifying mechanisms of progression and new therapeutic targets. Members of the transient receptor potential (TRP) family of cation-selective ion channels are candidates for mediating stress signals, since different subunits transduce a variety of stimuli relevant in both normal and pathogenic physiology. We addressed this possibility for the TRP vanilloid-1 (TRPV1) subunit by comparing how the optic projection of Trpv1(-/-) mice and age-matched C57 controls responds to stress from elevated ocular pressure, the critical stressor in the most common optic neuropathy, glaucoma. Over a 5 week period of elevated pressure induced by microbead occlusion of ocular fluid, Trpv1(-/-) accelerated both degradation of axonal transport from retinal ganglion cells to the superior colliculus and degeneration of the axons themselves in the optic nerve. Ganglion cell body loss, which is normally later in progression, occurred in nasal sectors of Trpv1(-/-) but not C57 retina. Pharmacological antagonism of TRPV1 in rats similarly accelerated ganglion cell axonopathy. Elevated ocular pressure resulted in differences in spontaneous firing rate and action potential threshold current in Trpv1(-/-) ganglion cells compared with C57. In the absence of elevated pressure, ganglion cells in the two strains had similar firing patterns. Based on these data, we propose that TRPV1 may help neurons respond to disease-relevant stressors by enhancing activity necessary for axonal signaling.

Phospholamban is a Cation Selective Ion Channel

Phospholamban (PLN) is a small integral membrane protein, which is involved in the contractility of cardiac muscle by regulating intracellular calcium concentration of cardiac myocytes. PLN binds and inhibits in a yet unknown way the sarco/endoplasmic reticulum Ca2+-ATPase. When reconstituted in planar lipid bilayers PLN exhibits ion channel activity with a main low unitary conductance, a sub-conductance level and a long open/closed dwell time[1]. From the effect of non-electrolyte polymers on this unitary conductance we estimate a narrow pore with a diameter of ca. 2.2 A[2]. This value is similar to that reported for the central pore in the structure of the PLN pentamer[3]. Hence the PLN pentamer, which is in equilibrium with the monomer[4], is the most likely channel forming structure. Moreover, recent data on selectivity show that the channel follows the II Eisenmann sequence (Rb>Cs>K>Na>Li). This pattern reveals that the ion selectivity is regulated by the radius of the binding site and not by the ionic radius. Our results combined with computational data suggest the presence of a selectivity filter in the pentamer PLN.[1] S. Smeazzetto, I. Schroder, G. Thiel, M. R. Moncelli, Phys. Chem. Chem. Phys. 2011, 13, 12935.[2] S. Smeazzetto, A. Saponaro, H. S. Young, M. R. Moncelli, G. Thiel, PLoS One 2013, 8, e52744.[3] K. Oxenoid, J. Chou, Proc. Natl. Acad. Sci. USA 2005, 102, 10870.[4] R. L. Cornea, L. R. Jones, J. M. Autry, D. D. Thomas, Biochemistry. 1997, 36, 2960.The financial support from Ente Cassa di Risparmio di Firenze and Ministero dell'Istruzione, Universita e Ricerca (PRIN project) and from Loewe initiative SoftControl are gratefully acknowledged.

Molecular Dynamics Simulations Reveal the HIV-1 Vpu Transmembrane Protein to Form Stable Pentamers

The human immunodeficiency virus type I (HIV-1) Vpu protein is 81 residues long and has two cytoplasmic and one transmembrane (TM) helical domains. The TM domain oligomerizes to form a monovalent cation selective ion channel and facilitates viral release from host cells. Exactly how many TM domains oligomerize to form the pore is still not understood, with experimental studies indicating the existence of a variety of oligomerization states. In this study, molecular dynamics (MD) simulations were performed to investigate the propensity of the Vpu TM domain to exist in tetrameric, pentameric, and hexameric forms. Starting with an idealized α-helical representation of the TM domain, a thorough search for the possible orientations of the monomer units within each oligomeric form was carried out using replica-exchange MD simulations in an implicit membrane environment. Extensive simulations in a fully hydrated lipid bilayer environment on representative structures obtained from the above approach showed the pentamer to be the most stable oligomeric state, with interhelical van der Waals interactions being critical for stability of the pentamer. Atomic details of the factors responsible for stable pentamer structures are presented. The structural features of the pentamer models are consistent with existing experimental information on the ion channel activity, existence of a kink around the Ile17, and the location of tetherin binding residues. Ser23 is proposed to play an important role in ion channel activity of Vpu and possibly in virus propagation.

Human PIEZO1: Removing Inactivation

PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by ∼30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. The slope sensitivity of gating was the same for wild-type and mutants, indicating that the dimensional changes between the closed and open state are unaffected by the mutations. The unitary channel conductance was unchanged by mutations, so these sites are not associated with pore. DhPIEZO1 was reversibly inhibited by the peptide GsMTx4 that acted as a gating modifier. The channel kinetics were solved using complex stimulus waveforms and the data fit to a three-state loop in detailed balance. The reaction had two pressure-dependent rates, closed to open and inactivated to closed. Pressure sensitivity of the opening rate with no sensitivity of the closing rate means that the energy barrier between them is located near the open state. Mutant cycle analysis of inactivation showed that the two sites interacted strongly, even though they are postulated to be on opposite sides of the membrane.

TRPV3: a ‘more than skinny’ channel

The skin is our largest organ serving different tasks from barrier formation through somatosensing to hair development. Recently, members of the large trp (Transient Receptor Potential, TRP) gene family encoding proteins that form cation selective ion channel have been identified to play a crucial role in skin functions. Within the 28 different mammalian TRP channels, TRPV3 might be the most important member in the skin. This review gives an overview on functional properties of TRPV3 in skin physiology and in certain skin diseases.

Cation-permeable vacuolar ion channels in the moss Physcomitrella patens: a patch-clamp study

Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca2+ on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K+ (E K), indicating K+ selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at −80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na+ and K+ selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na+ influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca2+ revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca2+ activated SV channels, vacuolar Ca2+ inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca2+ was also determined. These channels were active even without Ca2+ (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca2+ on the cytoplasmic side. Apart from monovalent cations (K+ and Na+), SV channels were permeable to divalent cations (Ca2+ and Mg2+). Both monovalent and divalent cations passed through the channels in the same direction—from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.

Temperature Dependence of Gramicidin Channel Transport and Structure

The gramicidin dipeptide is a small, cation-selective ion channel. Recent experiments have indicated that gramicidin can conduct ions at elevated temperatures. Since gramicidin is an efficient proton conductor, it is possible that this channel may have applications in fuel cell technology. In this study, we examine the temperature dependence of gramicidin A channel transport and structure with molecular dynamics simulations. In particular, the potentials of mean force (PMFs) for potassium ion motion through the channel are computed at five temperatures in the range 300–360 K. The channel displays a decrease in the free energy barrier height as the temperature increases. In addition, the enthalpic and entropic components of the free energy are computed, indicating a substantial enthalpy–entropy compensation and a positive entropy change when the ion enters the channel. The positive entropy change results from a reduction in fluctuations of ion interaction energies in the pore relative to those in the bulk solvent. The overall dimeric channel structure is maintained at 360 K for time scales up to 100 ns. In addition, higher temperatures affect the distributions of hydrogen bonds at the dimer interface, conformations of the N-terminal domain that may block the pore, channel bending angles, and distances between dimers. These findings may be related to the gating of the channel, although no complete dimer dissociation events were observed in the simulations.

Gating Transitions in the Palm Domain of ASIC1a*

Acid-sensing ion channels (ASICs) are trimeric cation-selective proton-gated ion channels expressed in the central and peripheral nervous systems. The pore-forming transmembrane helices in these channels are linked by short loops to the palm domain in the extracellular region. Here, we explore the contribution to proton gating and desensitization of Glu-79 and Glu-416 in the palm domain of ASIC1a. Engineered Cys, Lys, and Gln substitutions at these positions shifted apparent proton affinity toward more acidic values. Double mutant cycle analysis indicated that Glu-79 and Glu-416 cooperatively facilitated pore opening in response to extracellular acidification. Channels bearing Cys at position 79 or 416 were irreversibly modified by thiol-reactive reagents in a state-dependent manner. Glu-79 and Glu-416 are located in β-strands 1 and 12, respectively. The covalent modification by (2-(trimethylammonium)ethyl) methanethiosulfonate bromide of Cys at position 79 impacted conformational changes associated with pore closing during desensitization, whereas the modification of Cys at position 416 affected conformational changes associated with proton gating. These results suggest that β-strands 1 and 12 contribute antagonistically to activation and desensitization of ASIC1a. Site-directed mutagenesis experiments indicated that the lower palm domain contracts in response to extracellular acidification. Taken together, our studies suggest that the lower palm domain mediates conformational movements that drive pore opening and closing events.

Conductance of Ideally Cation-Selective Ion Channel Depends on Anion Type

Gramicidin A (gA) is a cation selective ion channel that has been used in many biophysical studies of lipid bilayers, in particular for investigations of lipid-protein interactions [1, 2] and membrane electrostatics [3]. In addition, it was found that ionic interactions with neutral lipid membranes also affect the kinetics of gA channels [4]. Here we report measurements of gA ion-channels for a series of sodium and potassium salts that show an anion-dependence of gA conductance. We find that gA conductance varies significantly with the anion type with ClO4 and SCN producing distinctly larger conductance values than Cl, F, and H2PO4. These results can provide new insights into ion-lipid membrane interactions and ion channel functions in general. [1] Andersen et al., Annu. Rev. Biophys. Biomol. Struct., 1996, [2] Lunbaek et al., PNAS 2010, [3] Rostovtseva et al. Biophys. J. 1998, [4] Rostovtseva et al. Biophys. J. 2008.

An Acidic Ring in the Palm Domain of ASIC1a Facilitates Pore opening

Acid-sensing ion channels (ASICs) are neuronal cation selective proton-gated ion channels that contribute to nociception, mechanosensation, synaptic plasticity, learning and memory, and fear conditioning. ASIC subunits have two transmembrane helices, intracellular N- and C- termini, and a large extracellular region organized in discrete domains named the thumb, finger, palm, beta-ball and knuckle. The palm domain is directly connected to the pore-forming transmembrane helices. We combined site-directed mutagenesis and electrophysiology to examine the contribution of residues Glu79 and Glu416 to ASIC1a proton-gating and desensitization. These acidic residues are located in the core of the palm domain forming a ring-like structure. Individual substitution at these positions to Cys, Lys or Gln shifted the apparent proton affinity of ASIC1a approximately one pH unit toward more acidic values. A similar shift in apparent proton affinity was observed in channels bearing substitutions at both positions, indicating that Glu79 and Glu416 cooperatively contribute to proton-gating. E79C and E416C mutants were modified by MTSET in the closed, but not in the desensitized state. MTSET-modification of E79C increased the magnitude of the response to protons by approximately six-fold and slowed desensitization by ten-fold, while MTSET-modification of E416C only reduced the response to protons nearly 50 percent. Ala mutagenesis at neighboring positions of Glu79 indicates that the covalently modified side chain of Cys at position 79 became closer to residues in the thumb and palm domains during channel gating. Our results suggest that Glu79 and Glu416 facilitate pore opening, and that the palm domain experiences a rotation during proton-dependent activation and desensitization.

Bulky Substitutions in the Thumb Domain of ASIC1a Restrict Proton-Gating

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation selective ion channels expressed in the nervous system. Each monomer consists of two transmembrane helices with short cytoplasmic tails and a large extracellular region organized in distinct domains named the thumb, finger, knuckle, β-ball, and palm. In this report we investigated the contribution to mouse ASIC1a activation and desensitization of three pairs of acidic residues, Glu238-Asp345, Asp237-Asp349 and Glu219-Asp407. Residues in each pair are located in close proximity and together form an acidic cluster at the interface between the thumb and β-ball domains. We examined the reactivity toward methanethiosulfonate (MTS) reagents of channels carrying individual Cys substitutions at these positions. MTSET-modification of D345C channels shifted the pH of half-maximal activation (pH50) from 6.23±0.06 to 4.48±0.08, and increased the desensitization rate. A similar effect was observed with other positively-charged MTS reagents such as MTSMT, MTSPT and MTSEA, as well as with MTSES, a negatively-charged reagent. Moreover, Lys substitution at position 345 shifted pH50 toward more acidic values. Remarkably, Lys substitution at positions 219, 237 or 238 did not significantly change the pH50, suggesting that acid residues at these positions do not substantially contribute to proton activation. Our results indicate that the introduction of a bulky moiety at position 345 restricts conformational movements required for proton-gating, suggesting that the thumb-β-ball interface undergoes dynamic rearrangements during activation.

Antihypertensive Antagonists of L-Type Calcium Channel Exert Enhancement of Alzheimer's Aβ Peptides Effect on Cells

The Alzheimer's Aβ peptides are shown to increase the cytosolic calcium concentrations of cells in culture. Such an alteration may cause a variety of secondary effects, leading to cell degeneration and reduced cell culture growth. Cytosolic calcium concentration can be increased by the formation of cation-selective ion channels by the Aβ peptides. This property is well documented and has been extensively studied both in artificial membrane systems as well as in living cells. Here we show that L-type calcium channel antagonists, commonly used in the treatment of hypertension and ischemic heart disease, intensify the capacity of Aβ peptides to increase cytosolic calcium concentrations and consequently enhance the cytotoxicity of the Aβ peptides. Calcium imaging experiments were conducted with the fluorescent indicator Fura-2 to measure at different periods of times the levels of cytosolic calcium during exposure to Aβ peptides and to the L-type calcium channel antagonists. Two different 1,4-dihydropyridines (nifedipine, and nitrendipine), and Diltiazem, a structurally unrelated Ca2+-channel antagonist belonging to the phenylalkylamine class were used and found to exert significant excitatory effects on the toxicity of Aβ. The reduced toxicity observed in solutions of combinations of Aβ40 and Aβ42 is also reversed by L-type calcium channel antagonists. The toxicity of Aβ peptides was weighed by quantifying the growth of cultures of PC12 cells using the colorimetric assay XTT and by measuring the release of LDH to the media. The enhancement of the Aβ peptides toxicity by L-type calcium channel antagonists can be constrained by specific Aβ ion channel blockers suggesting the mediation of the ion channel property of Aβ peptides. The relevance of our finding derives from the widespread clinical use of L-type calcium channel antagonists in the treatment of hypertension and angina pectoris.