Slow Action Potentials Research Articles

Axonal ER Ca2+ Release Selectively Enhances Activity-Independent Glutamate Release in a Huntington Disease Model.

Action potential-independent (miniature) neurotransmission occurs at all chemical synapses, but remains poorly understood, particularly in pathological contexts. Axonal endoplasmic reticulum (ER) Ca2+ stores are thought to influence miniature neurotransmission, and aberrant ER Ca2+ handling is implicated in progression of Huntington disease (HD). Here, we report elevated miniature excitatory postsynaptic current (mEPSC) frequencies in recordings from YAC128 mouse (HD-model) neurons (from cortical cultures and striatum-containing brain slices - both from male and female animals). Pharmacological experiments suggest this is mediated indirectly by enhanced tonic ER Ca2+ release. Calcium imaging, using an axon-localized sensor, revealed slow action potential (AP)-independent ER Ca2+ release waves in both YAC128 and wild-type cultures. These Ca2+ waves occurred at similar frequencies in both genotypes, but spread less extensively and were of lower amplitude in YAC128 axons, consistent with axonal ER Ca2+ store depletion. Surprisingly, basal cytosolic Ca2+ levels were lower in YAC128 boutons and YAC128 mEPSCs were less sensitive to intracellular Ca2+ chelation. Together, these data suggest elevated miniature glutamate release in YAC128 cultures is associated with axonal ER Ca2+ depletion but not directly mediated by ER Ca2+ release into the cytoplasm. In contrast to increased mEPSC frequencies, cultured YAC128 cortical neurons showed less frequent AP-dependent (spontaneous) Ca2+ events in soma and axons, although evoked glutamate release detected by iGluSnFR in brain slices was similar between genotypes. Our results indicate axonal ER dysfunction selectively elevates miniature glutamate release from cortical terminals in HD. This, together with reduced spontaneous cortical neuron firing, may cause a shift from activity-dependent to -independent glutamate release in HD, with potential implications for fidelity and plasticity of cortical excitatory signaling.Significance statementMiniature neurotransmitter release persists at all chemical neuronal synapses in the absence of action potential firing but remains poorly understood, particularly in disease states. We show enhanced miniature glutamate release from cortical neurons in the YAC128 mouse Huntington disease model. This effect is mediated by axonal ER Ca2+ store depletion, but is not obviously due to elevated ER-to-cytosol Ca2+ release. Conversely, YAC128 cortical pyramidal neurons fired fewer action potentials and evoked cortical glutamate release was similar between WT an YAC128 preparations, indicating axonal ER depletion selectively enhances miniature glutamate release in YAC128 mice. These results extend our understanding of action potential independent neurotransmission and highlight a potential involvement of elevated miniature glutamate release in Huntington disease pathology.

Electrophysiological Recordings from Embryonic Mouse Motoneurons Cultured on Electrospun Poly-Lactic Acid (PLA) and Polypyrrole-Coated PLA Scaffolds.

Background:Biomaterials used as cell growth stimulants should be able to provide adequate cell adhesion with no alteration in cell function. In this work, we developed a 3D model of mouse spinal cord motoneurons on scaffolds composed of electrospun PLA fibers and plasma-polymerized PPy-coated PLA fibers. Methods:The functionality of the cultured motoneurons was assessed by evaluating both the electrophysiological response (i.e., the whole-cell Na+ and K+ currents and the firing of action potentials) and also the expression of the VAChaT by immunostaining techniques. While the expression of the VAChaT was confirmed on motoneurons cultured on the fibrous scaffolds, the electrophysiological responses indicated Na+ and K+ currents with lower amplitude and slower action potentials when compared to the response recorded from spinal cord motoneurons cultured on Poly-DL-Ornithine/Laminin- and plasma-polymerized PPy-coated coverslips.Results:From a morphological viewpoint, motoneurons cultured on PLA and PPy-coated PLA scaffolds did not show the development of dendritic and/or axonal processes, which were satisfactorily observed in the bidimensional cultures.Conclusion:We hypothesize that the apparently limited development of dendritic and/or axonal processes could produce a deleterious effect on the electrophysiological response of the cells, which might be due to the limited growth surface available in the fibrous scaffolds and/or to an undesired effect of the purification process.

Role of LVA Ca channels in norepinephrine-induced automatic activity in rat pulmonary vein myocardial sleeve

Ectopic foci in pulmonary veins (PV) are involved in the onset of atrial fibrillation. Norepinephrine (NE) induces in rat PV cardiomyocytes (CM) an automatic activity in saw-tooth pattern (catecholaminergic automatic activity or CAA), consisting of bursts of slow action potentials which depends on Ca (upstroke) and Na (inter burst) channels. We have previously shown the co-existence in rat PVCM, of a fast LVA (FLVA) non selective TTX-sensitive cationic channel and of T-type Ca channels. To evaluate in male Wistar rat PV myocardial sleeve the contribution of these channels to triggering NE-induced ectopic foci. Whole-cell currents were studied at room temperature in enzymatically isolated PVCM. Mechanical activity of PV rings was recorded in an organ bath at 37 °C and CAA was induced by application of 10 μM NE. The FLVA Ca current was increased by the Cav3.x blockers TTA-A2 (10-100 nM, n = 11) and NiCl2 (40 μM, n = 9) whereas a large LVA Ca current, present in 10 out of 80 PVCM, was partially reduced by 100 nM TTA-A2. Study of K currents revealed the presence of an inward rectifying current sensitive to TRAM-34 (1 μM, n = 6), a blocker of the Ca-activated K channel SK4. In PV rings, TTA-A2 (1-5 μM) decreased CAA incidence. Total arrhythmia duration, measured over the last 10 min of a 30 min long drug application, was reduced as well as burst duration and the number of spontaneous twitches within a burst. On the other hand, 40 μM NiCl2 or 1-2 μM TRAM-34 increased total arrhythmia duration by decreasing the interval between bursts (no significant effect on the burst characteristics). Only NiCl2 had a significant negative inotropic effect (∼80% inhibition of L-type calcium current). Our results suggest that Ca influx through Cav3.1 channels contribute to triggering bursts of automatic activity and that Cav3.2 Ca channels, through activation of SK4 channels, are involved in the regulation of the interval between bursts.

Low voltage-activated channels in rat pulmonary vein cardiomyocytes: coexistence of a non-selective cationic channel and of T-type Ca channels.

In rat pulmonary vein (PV) cardiomyocytes (CM), norepinephrine (NE) induces an automatic activity consisting of bursts of slow action potentials which depend on Ca2+ (upstroke) and Na+ (inter-burst) channels. Our objective was to characterize low voltage-activated (LVA) currents in rat PVCM susceptible to trigger this activity. Whole-cell ICa (5 mM Ca2+) was recorded from - 100 mV with classical Na+- and K+-free solutions. A fast LVA ICa (FLVA-ICa), present in ≈ 56% of PVCM between ~ - 50 to - 20 mV, was blocked by 10 μM TTX and markedly increased by addition of NaCl (1 or 3 mM) or KCl (5 or 10 mM). Permeability ratios P'Ca/PNa and P'Ca/PK calculated for bi-ionic conditions were respectively 2.25 ± 0.51 and 1.88 ± 0.25, and not different from a value of 2. FLVA-ICa was increased by 10 μM NE and 300 nM BayK8644, decreased by 5 μM nifedipine but not blocked by ranolazine (10 μM). NiCl2 (40 μM) and TTA-A2 (10 or 100 nM) increased FLVA-ICa. Similar results were obtained in left atrial (LA) CM. Neither Ba2+ nor Sr2+ alone could permeate the FLVA channel or block Ca2+ influx but revealed a large slower activating and inactivating LVA Ca2+ current (SLVA-ICa), present in 10 out of 80 PVCM, absent in LACM, and partially inhibited by 100 nM TTA-A2. Therefore, the ionic channel underlying FLVA-ICa is likely a fast voltage-gated non-selective channel with a dihydropyridine binding site. SLVA-ICa might correspond to Ca2+ influx through Cav3.x channels and contribute to triggering NE-induced automatic activity in the PV myocardial sleeve.

Structure and function of the nervous system in nectophores of the siphonophore Nanomia bijuga.

Although the bell-shaped nectophores of the siphonophore Nanomia bijuga are clearly specialized for locomotion, their complex neuroanatomy described here testifies to multiple subsidiary functions. These include secretion, by the extensively innervated 'flask cells' located around the bell margin, and protection, by the numerous nematocytes that line the nectophore's exposed ridges. The main nerve complex consists of a nerve ring at the base of the bell, an adjacent column-shaped matrix plus two associated nerve projections. At the top of the nectophore the upper nerve tract appears to have a sensory role; on the lower surface a second nerve tract provides a motor input connecting the nectophore with the rest of the colony via a cluster of nerve cells at the stem. N. bijuga is capable of both forward and backward jet-propelled swimming. During backwards swimming the water jet is redirected by the contraction of the Claus' muscle system, part of the muscular velum that fringes the bell aperture. Contractions can be elicited by electrical stimulation of the nectophore surface, even when both upper and lower nerve tracts have been destroyed. Epithelial impulses elicited there, generate slow potentials and action potentials in the velum musculature. Slow potentials arise at different sites around the bell margin and give rise to action potentials in contracting Claus' muscle fibres. A synaptic rather than an electrotonic model more readily accounts for the time course of the slow potentials. During backward swimming, isometrically contracting muscle fibres in the endoderm provide the Claus' fibres with an immobile base.

Electrophysiological effects of ischemia/reperfusion (I/R) on pacemaker cells in guinea-pig left ventricular outflow tract and the effects of antiarrhythmic drugs treatment

To investigate the electrophysiological effects of ischemia/reperfusion (I/R) on the spontaneous slow response action potentials in guinea-pig left ventricular outflow tract and the effects of antiarrhythmic drugs onI/R. The action potentials of pacemaker cells in guinea-pig left ventricular outflow tract were recorded by conventional intracellular microelectrode technique. The influences of ischemia(I) 10 min, reperfusion(R) 2 min, and R 15min on the spontaneous slow response potentials were investigated. The effects of lidocaine, propafenone, amiodarone, verapamil, adenosine, and sodium nitroprusside (SNP) on I/R were also studied. Electrophysiological parameters were examined:velocity of diastolic depolarization(VDD), rate of pacemaker firing(RPF), maximal diastolic potential(MDP), maximal rate of depolarization(Vmax), amplitude of action potential(APA), 50% and 90% of duration of action potential(APD50 and APD90). ①In I 10 min group, the values of VDD, RPF, Vmax and APA were decreased markedly compared with control group (P<0.05, P<0.01).In R 2 min group, VDD and RPF were increased significantly(P<0.01), MDP was increased notably(P<0.05), APD50 and APD90 were shortened significantly compared with I 10 min and control group. Vmax was increased markedly vs control group(P<0.05). APA was decreased notably vs I 10 min group (P<0.05), but was increased markedly vs control group(P<0.05). In R 15 min group, the action potentials recovered gradually to the levels of control group. ② Compared with I 10 min/R 2 min group, 1 μmol/L lidocaine, 10 μmol/L propafenone, 1 μmol/L amiodarone, 1 μmol/L verapamil, 50 μmol/L adenosine, and 10 μmol/L SNP decreased VDD and RPF significantly. I/R injury can trigger abnormal spontaneous activities of guinea-pig left ventricular outflow tract.The electrophysiological effects of I/R injury on left ventricular outflow tract can be treated by antiarrhythmic drugs.

Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navβ4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction.

Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs). Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navβ4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navβ4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have distinct biophysical properties that may differentially contribute to regulating neuronal excitability.

Aging Alters cAMP Signaling and Membrane-Delimited Regulation of if in Sinoatrial Myocytes

We recently demonstrated that age-dependent declines in intrinsic and maximum heart rates are associated with slower action potential (AP) firing rate and a hyperpolarizing shift in the midpoint activation voltage (V1/2) of the funny current (If) in isolated sinoatrial myocytes (SAMs). Given that cAMP is an important intracellular modulator in SAMs, we hypothesized that reduced cAMP concentration may contribute to the slower APs and shifted If in aged SAMs. To test this hypothesis, we applied a saturating concentration of cAMP via the patch pipette in whole cell voltage-clamp and current clamp experiments in acutely isolated SAMs from young and old mice. Here, we show that this exogenous cAMP completely reversed the age-dependent changes in both AP firing rate and If in SAMs. In contrast, maximal stimulation of endogenous cAMP production, via co-application of the PDE inhibitor, IBMX and the adenylyl cyclase activator, forskolin, did not rescue either AP firing rate or If in aged SAMs. These data indicate that a deficit in cAMP production and/or responsiveness may contribute to the age-dependent changes in SAM excitability. To further determine how cAMP regulates If in aged versus young SAMs, we compared If activation in excised inside-out membrane patches from SAMs from young and old mice. Surprisingly, we found that the age-dependent hyperpolarized shift in the V1/2 of If persisted in the cell-free patches, indicating that soluble cAMP alone cannot account for the shifted If in aged SAMs. Rather, aging appears to alter an additional, membrane-associated factor that regulates If in SAMs. Future work will focus on resolving the underlying mechanism(s) responsible for the age-dependent shift in the V1/2 of If, and its relationship to AP generation in SAMs.

HERG 1b is critical for human cardiac repolarization.

The human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying IKr, a repolarizing current in the heart. Mutations in KCNH2 or pharmacological agents that reduce IKr slow action potential (AP) repolarization and can trigger cardiac arrhythmias associated with long QT syndrome. Two channel-forming subunits encoded by KCNH2 (hERG 1a and 1b) are expressed in cardiac tissue. In heterologous expression systems, these subunits avidly coassemble and exhibit biophysical and pharmacological properties distinct from those of homomeric hERG 1a channels. Despite these findings, adoption of hERG 1a/1b heteromeric channels as a model for cardiac IKr has been hampered by the lack of evidence for a direct functional role for the 1b subunit in native tissue. In this study, we measured IKr and APs at physiological temperature in cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs). We found that specific knockdown of the 1b subunit using shRNA caused reductions in 1b mRNA, 1b protein levels, and IKr magnitude by roughly one-half. AP duration was increased and AP variability was enhanced relative to controls. Early afterdepolarizations, considered cellular substrates for arrhythmia, were also observed in cells with reduced 1b expression. Similar behavior was elicited when channels were effectively converted from heteromers to 1a homomers by expressing a fragment corresponding to the 1a-specific N-terminal Per-Arnt-Sim domain, which is omitted from hERG 1b by alternate transcription. These findings establish that hERG 1b is critical for normal repolarization and that loss of 1b is proarrhythmic in human cardiac cells.

Antiarrhythmic effects of dehydroevodiamine in isolated human myocardium and cardiomyocytes

Ethnopharmacological relevanceDehydroevodiamine alkaloid (DeHE), a bioactive component of the Chinese herbal medicine Wu-Chu-Yu (Evodiae frutus), exerted antiarrhythmic effect in guinea-pig ventricular myocytes. We further characterize the electromechanical effects of DeHE in the human atrial and ventricular tissues obtained from hearts of patients undergoing corrective cardiac surgery or heart transplantation. Materials and methodsThe transmembrane potentials of human myocardia were recorded with a traditional microelectrode technique while sarcolemmal Na+ and Ca2+ currents in single human cardiomyocytes were measured by a whole-cell patch-clamp technique. The intracellular pH (pHi) and Na+–H+ exchanger (NHE) activity were determined using BCECF-fluorescence in human atria. ResultsIn human atria, DeHE (0.1–0.3μM) depressed upstroke velocity, amplitude of action potential, and contractile force, both in slow and fast response action potential. Moreover, the similar depressant effects of DeHE were found in human ventricular myocardium. Both in isolated human atrial and ventricular myocytes, DeHE (0.1–1μM) reversibly, concentration-dependently decreased the Na+ and Ca2+currents. Moreover, DeHE (0.1 and 0.3μM) suppressed delayed afterdepolarizations and aftercontractions, induced by epinephrine and high [Ca2+]o in atria. In human ventricular myocardium, the strophanthidin-induced triggered activities were attenuated by pretreating DeHE (0.3μM). The resting pHi and NHE activity were also significantly increased by DeHE (0.1–0.3μM). ConclusionsWe concluded for the first time that, in the human hearts, DeHE could antagonize triggered arrhythmias induced by cardiotonic agents through a general reduction of the Na+ and Ca2+ inward currents, while increase of resting pHi and NHE activity.

The recovery from AP adaptation in sensorineural hearing loss

Conclusions: An abnormally slower action potential (AP) recovery from adaptation (decreased recovery, dR) was characteristically detected in many ears with sensorineural hearing loss (SNHL) but not Meniere's disease and idiopathic sudden SNHL. We assumed that this abnormal AP recovery from adaptation was attributed to an imbalance in the distributions of auditory neurons with high and low spontaneous firing rates. The significant difference of initial hearing level between dR and normal AP recovery groups (nR) was assumed to partially result from AP recovery being determined by the inner hair cell synapse, and not from outer hair cells. Objective: This study aimed to detect the AP recovery pattern in SNHL. Methods: Electrocochleography (ECochG) was performed transtympanically in 30 patients with SNHL. AP recovery was measured by a paired click stimulation paradigm as a function of inter-click intervals from 5 to 100 ms. Results: The high prevalence of dR (9 of 30 ears) appears to be a characteristic ECochG finding in SNHL. Initial hearing level differed significantly between dR and nR groups.

ORM‐10103, a novel specific inhibitor of the Na+/Ca2+ exchanger, decreases early and delayed afterdepolarizations in the canine heart

At present there are no small molecule inhibitors that show strong selectivity for the Na(+) /Ca(2+) exchanger (NCX). Hence, we studied the electrophysiological effects of acute administration of ORM-10103, a new NCX inhibitor, on the NCX and L-type Ca(2+) currents and on the formation of early and delayed afterdepolarizations. Ion currents were recorded by using a voltage clamp technique in canine single ventricular cells, and action potentials were obtained from canine and guinea pig ventricular preparations with the use of microelectrodes. ORM-10103 significantly reduced both the inward and outward NCX currents. Even at a high concentration (10 μM), ORM-10103 did not significantly change the L-type Ca(2+) current or the maximum rate of depolarization (dV/dtmax ), indicative of the fast inward Na(+) current. At 10 μM ORM-10103 did not affect the amplitude or the dV/dtmax of the slow response action potentials recorded from guinea pig papillary muscles, which suggests it had no effect on the L-type Ca(2+) current. ORM-10103 did not influence the Na(+) /K(+) pump or the main K(+) currents of canine ventricular myocytes, except the rapid delayed rectifier K(+) current, which was slightly diminished by the drug at 3 μM. The amplitudes of pharmacologically- induced early and delayed afterdepolarizations were significantly decreased by ORM-10103 (3 and 10 μM) in a concentration-dependent manner. ORM-10103 is a selective inhibitor of the NCX current and can abolish triggered arrhythmias. Hence, it has the potential to be used to prevent arrhythmogenic events.

Genetic engineering of somatic cells to study and improve cardiac function

To demonstrate the utility of genetically engineered excitable cells for studies of basic electrophysiology and cardiac cell therapy. 'Zig-zag' networks of neonatal rat ventricular myocytes (NRVMs) were micropatterned onto thin elastomeric films to mimic the slow action potential (AP) conduction found in fibrotic myocardium. Addition of genetically engineered excitable human embryonic kidney cells (HEK-293 cells) ('Ex-293' cells stably expressing Kir2.1, Na(v)1.5, and Cx43 channels) increased both cardiac conduction velocity by 370% and twitch force amplitude by 64%. Furthermore, we stably expressed mutant Na(v)1.5 [A1924T (fast sodium channel mutant (substitution of alanine by threonine at amino acid 1924)] channels with hyperpolarized steady-state activation and showed that, despite a 71.6% reduction in peak I(Na), these cells propagated APs at the same velocity as the wild-type Na(v)1.5-expressing Ex-293 cells. Stable expression of Ca(v)3.3 (T-type voltage-gated calcium) channels in Ex-293 cells (to generate an 'ExCa-293' line) significantly increased their AP duration and reduced repolarization gradients in cocultures of these cells and NRVMs. Additional expression of an optogenetic construct [ChIEF (light-gated Channelrhodopsin mutant)]enabled light-based control of AP firing in ExCa-293 cells. We show that, despite being non-contractile, genetically engineered excitable cells can significantly improve both electrical and mechanical function of engineered cardiac tissues in vitro. We further demonstrate the utility of engineered cells for tissue-level studies of basic electrophysiology and cardiac channelopathies. In the future, this novel platform could be utilized in the high-throughput design of new genetically encoded indicators of cell electrical function, validation, and improvement of computer models of AP conduction, and development of novel engineered somatic cell therapies for the treatment of cardiac infarction and arrhythmias.

RGS2 overexpression or Gi inhibition rescues the impaired PKA signaling and slow AP firing of cultured adult rabbit pacemaker cells

Freshly isolated adult rabbit sinoatrial node cells (f-SANC) are an excellent model for studies of autonomic signaling, but are not amenable to genetic manipulation. We have developed and characterized a stable cultured rabbit SANC (c-SANC) model that is suitable for genetic manipulation to probe mechanisms of spontaneous action potential (AP) firing.After 48h in culture, c-SANC generate stable, rhythmic APs at 34±0.5°C, at a rate that is 50% less than f-SANC. In c- vs. f-SANC: AP duration is prolonged; phosphorylation of phospholamban at Ser16 and type2 ryanodine receptor (RyR2) at Ser2809 are reduced; and the level of type2 regulator of G-protein signaling (RGS2), that facilitates adenylyl cyclases/cAMP/protein kinase A (PKA) via Gi inhibition, is substantially reduced. Consistent with the interpretation that cAMP/PKA signaling becomes impaired in c-SANC, acute β-adrenergic receptor stimulation increases phospholamban and RyR2 phosphorylation, enhances RGS2-labeling density, and accelerates the AP firing rate to the similar maximum in c- and f-SANC. Specific PKA inhibition completely inhibits all β-adrenergic receptor effects. Adv-RGS2 infection, or pertussis toxin treatment to disable Gi-signaling, each partially rescues the c-SANC spontaneous AP firing rate.Thus, a Gi-dependent reduction in PKA-dependent protein phosphorylation, including that of Ca2+ cycling proteins, reduces the spontaneous AP firing rate of c-SANC, and can be reversed by genetic or pharmacologic manipulation of PKA signaling.

What does type A mean to a pyramidal cell?

Potassium channels powerfully control neuronal excitability and exhibit great molecular diversity. Despite this diversity, classification of voltage-gated potassium currents is often simplified into a distinction between a rapidly activating, transient A-type current and a slowly activating and inactivating delayed rectifier (DR) current. However, individual neurons express multiple potassium channel subunits from several families and knowing the functional roles of each of these subunits is essential to understanding neural computations and pathophysiology. As an example, components of the A-type current in neocortical pyramidal cells are generated by Kv4.2, Kv4.3 and Kv1.4 α subunits (Norris & Nerbonne, 2010). In this issue of The Journal of Physiology, Carrasquillo et al. (2012) address the functional roles of each of these subunit types with slice recordings from visual cortex of mice where Kv4.2 (Kv4.2−/−), Kv4.3 (Kv4.3−/−), or Kv1.4 (Kv1.4−/−) subunit protein was eliminated. The consequences of null mutations differed between the three subunits, suggesting distinct roles in regulating membrane properties (Fig. 1). Figure 1 Functional roles of Kv4.2, Kv4.3 and Kv1.4 subunits in neocortical pyramidal neurons (determined by Carrasquillo et al. 2012) In pyramidal neurons from Kv4.2−/− mice, input resistance was increased, the current threshold for action potential (AP) generation was reduced, APs were broader and rebound firing in response to prolonged hyperpolarization was increased. Cells from Kv4.3−/− mice also exhibited increased spike width, but input resistance, current threshold and rebound firing were unchanged. These data suggest greater importance of Kv4.2 at subthreshold voltages, perhaps due to a more hyperpolarized activation range. Elimination of either Kv4.2 or Kv4.3 resulted in faster firing than wild-type neurons but changes in the Kv4.3−/− cells were not significant until larger current injections. In contrast, the Kv4.2−/− cells exhibited greater effects to small current injections than larger ones. The reduced effect of Kv4.2−/− on firing in response to large stimuli may reflect slower recovery from inactivation of Kv4.2 channels. The effects of removing Kv4.2 and Kv4.3 were generally additive, although spike doublets were observed in some of the double cells lacking both Kv4.2 and Kv4.3. These findings allow consideration of whether Kv4 channels normally exist as homomeric or heteromeric channels in pyramidal cells. Previous studies suggested that Kv4.2 and Kv4.3 α subunits form heteromultimers but the present results show differential effects of removal of Kv4.2 and Kv4.3, suggesting that not all channels contain both subunits or that the heteromeric channels have different properties than homomeric channels. It remains to be determined whether the differential effects of subunit removal reflect the elimination of homomeric channels with different biophysical properties, replacement of heteromeric Kv4.2/Kv4.3 channels with homomers, or changes in channel targeting as a result of subunit removal. Carrasquillo et al. (2012) found no significant differences in input resistance, current thresholds, or firing rate in the Kv1.4 −/− mouse; however, pharmacological block of Kv4-mediated A currents with Ba2+ unmasked contributions of Kv1.4 to input resistance, AP current threshold, and repetitive firing. Interestingly, APs were actually narrower in Kv1.4−/− cells, apparently due to a compensatory increase in Kv4.2 channels. It is unclear if upregulation of Kv4.3 or Kv1.4 subunits also occurs with Kv4.2 elimination, but previous studies suggest functional compensation by the DR current. For example, Yuan et al. (2005) reported the absence of A-type current and slower AP repolarization with acute reduction of Kv4.2 channels. In contrast, in most cells from animals lacking Kv4.2 from birth the A-type current was absent yet AP repolarization was normal (Nerbonne et al. 2008). An increased DR current was observed in cells from the Kv4.2−/− animals and this was apparently able to fully compensate for AP repolarization. It was subsequently observed that partial block of the DR with TEA in Kv4.2−/− cells unmasked A-type currents mediated by Kv4.3 and Kv1.4 subunits (confirmed with Kv4.3−/− and Kv1.4−/− animals: Norris & Nerbonne, 2010). Collectively, these observations suggest that long-term elimination of an A-type current component may lead to compensatory changes in other A-type channels or other types of potassium channel. This obviously complicates the interpretation of findings in –/– animals and underscores the need to understand the rules by which such compensatory changes may occur. While the present results concentrate on somatodendritic channels, functional roles of A-type channels depend upon the precise location of their expression. It is not clear to what degree channel localization is altered in the –/– animals. The effectiveness of functional compensation may also vary in different cellular compartments. Still to be resolved is how much natural variation exists in the expression of Kv1.4, Kv4.2, and Kv4.3 and how this relates to known differences in active properties of pyramidal cell populations defined by layer, gene expression, or projection targets (cf. Hattox & Nelson, 2007). With this comprehensive series of papers, Nerbonne and colleagues show us just how complex the molecular basis and functional consequences can be for the seemingly simple type A current.

Targeting ion channels for the treatment of gastrointestinal motility disorders.

Gastrointestinal (GI) functional and motility disorders are highly prevalent and responsible for long-term morbidity and sometimes mortality in the affected patients. It is estimated that one in three persons has a GI functional or motility disorder. However, diagnosis and treatment of these widespread conditions remains challenging. This partly stems from the multisystem pathophysiology, including processing abnormalities in the central and peripheral (enteric) nervous systems and motor dysfunction in the GI wall. Interstitial cells of Cajal (ICCs) are central to the generation and propagation of the cyclical electrical activity and smooth muscle cells (SMCs) are responsible for electromechanical coupling. In these and other excitable cells voltage-sensitive ion channels (VSICs) are the main molecular units that generate and regulate electrical activity. Thus, VSICs are potential targets for intervention in GI motility disorders. Research in this area has flourished with advances in the experimental methods in molecular and structural biology and electrophysiology. However, our understanding of the molecular mechanisms responsible for the complex and variable electrical behavior of ICCs and SMCs remains incomplete. In this review, we focus on the slow waves and action potentials in ICCs and SMCs. We describe the constituent VSICs, which include voltage-gated sodium (Na(V)), calcium (Ca(V)), potassium (K(V), K(Ca)), chloride (Cl(-)) and nonselective ion channels (transient receptor potentials [TRPs]). VSICs have significant structural homology and common functional mechanisms. We outline the approaches and limitations and provide examples of targeting VSICs at the pores, voltage sensors and alternatively spliced sites. Rational drug design can come from an integrated view of the structure and mechanisms of gating and activation by voltage or mechanical stress.

Serial electrophysiological changes in uraemic patients with tetrodotoxin intoxication

Tetrodotoxin (TTX) is a well-known biological neurotoxin, predominantly isolated from the viscera of puffer fish. TTX binds to the voltage-gated sodium channels and reduces the membrane excitability of vital tissues (Narahashi et al., 1967). As a result, paraesthesia, paralysis, respiratory failure and, eventually, coma with absence of electrical brain activities can appear depending on the amounts of ingested toxin. There are only a few reports about the electrophysiological studies of peripheral nerves in TTX intoxication because of the rarity of this intoxication and rapid alteration of clinical and electrophysiological status. We conducted serial nerve-conduction studies (NCSs) in two uraemic patients with acute TTX intoxication during the recovery period and obtained the strength–duration curves with nerve-excitability test at each visit. A brief case summary is as follows: Patient #1 was a 38-year-old man, who received peritoneal dialysis with end-stage renal disease for 1 year. He was referred to our hospital due to rapidly progressive quadriparesis. Three days before admission, he ate cooked puffer fish for dinner. On the next morning, he felt dizziness and peri-oral tingling sensation. On neurologic examination, he showed symmetric muscle weakness of Medical Research Council grade 2. Although he complained of mild dyspnoea, he did not need mechanical ventilation. On the 5th day of intoxication, an NCS on the median nerve revealed slow conduction velocity (30 m s ) but normal compound muscle action potential (CMAP) amplitude (8 mV). Serum blood urea nitrogen (BUN) and creatinine (Cr) were 99 and 14.9 mg dl , respectively. Serum potassium level was normal (3.9 mEq l ). His neurologic deficits slowly improved and disappeared at 14 days after ingestion. In the follow-up NCS performed on the 17th day, CMAP was increased in amplitude (11 mV) and conduction velocity demonstrated improvement (51 m s ) over the previous value. A nerve-excitability test was performed. Recording electrodes were attached in the abductor pollicis brevis muscle and the median nerve at the wrist was stimulated. We set the initial stimulation duration to 0.05 ms of square wave, and stimulation intensities were serially increased with 1-mA intervals until visible muscle twitching was observed. We conducted the same procedure with each increment of 0.1 ms up to 1 ms to measure each threshold.

Simulation of the Undiseased Human Cardiac Ventricular Action Potential: Model Formulation and Experimental Validation

Cellular electrophysiology experiments, important for understanding cardiac arrhythmia mechanisms, are usually performed with channels expressed in non myocytes, or with non-human myocytes. Differences between cell types and species affect results. Thus, an accurate model for the undiseased human ventricular action potential (AP) which reproduces a broad range of physiological behaviors is needed. Such a model requires extensive experimental data, but essential elements have been unavailable. Here, we develop a human ventricular AP model using new undiseased human ventricular data: Ca2+ versus voltage dependent inactivation of L-type Ca2+ current (ICaL); kinetics for the transient outward, rapid delayed rectifier (IKr), Na+/Ca2+ exchange (INaCa), and inward rectifier currents; AP recordings at all physiological cycle lengths; and rate dependence and restitution of AP duration (APD) with and without a variety of specific channel blockers. Simulated APs reproduced the experimental AP morphology, APD rate dependence, and restitution. Using undiseased human mRNA and protein data, models for different transmural cell types were developed. Experiments for rate dependence of Ca2+ (including peak and decay) and intracellular sodium ([Na+]i) in undiseased human myocytes were quantitatively reproduced by the model. Early afterdepolarizations were induced by IKr block during slow pacing, and AP and Ca2+ alternans appeared at rates >200 bpm, as observed in the nonfailing human ventricle. Ca2+/calmodulin-dependent protein kinase II (CaMK) modulated rate dependence of Ca2+ cycling. INaCa linked Ca2+ alternation to AP alternans. CaMK suppression or SERCA upregulation eliminated alternans. Steady state APD rate dependence was caused primarily by changes in [Na+]i, via its modulation of the electrogenic Na+/K+ ATPase current. At fast pacing rates, late Na+ current and ICaL were also contributors. APD shortening during restitution was primarily dependent on reduced late Na+ and ICaL currents due to inactivation at short diastolic intervals, with additional contribution from elevated IKr due to incomplete deactivation.

Inhibitory Effects of Tiamulin on Contractile and Electrical Responses in Isolated Thoracic Aorta and Cardiac Muscle of Guinea-pigs

The inhibitory effect of tiamulin, an antibiotic produced by Pleurotus mutilis, on contractile and electrical responses in isolated thoracic aorta and cardiac muscle of guinea-pigs was studied. In the thoracic aorta, tiamulin with an IC50 of 9.7 x 10(-6) M inhibited sustained contractions induced by isosmotically added 60 mM KCl. The inhibitory effect of tiamulin on a Ca(2+)-induced contraction in a depolarized muscle was competitively antagonized by raising external Ca2+ concentration. Bay K 8644 (10(-7) M) antagonized tiamulin's inhibition of the Ca(2+)-induced contraction. Tiamulin (2 x 10(-5) M) decreased the elevated cytoplasmic Ca2+ level measured by the fura 2 AM method in the depolarized muscle. In high K(+)-isoprenaline-treated left atria, tiamulin (2 x 10(-5)-2 x 10(-4) M) produced negative inotropic effects. On the other hand in the membrane action potential of papillary muscles, tiamulin (2 x 10(-6)-2 x 10(-4) M) produced decreases in action potential and durations and 2 x 10(-4) M tiamulin depressed the slow response action potential in depolarized muscles. Tiamulin produced prolongations of the PR interval in ECG, negative chrono- and inotropic effects, and an increase in perfusion flow in guinea-pig isolated and perfused hearts. These effects of tiamulin on the aorta or cardiac muscle were similar to those of verapamil and nifedipine. These results suggest that both the inhibitory action of tiamulin on the high K(+)-induced contraction in the aorta and the negative inotropic effect of tiamulin on the cardiac muscle are due to an inhibition of Ca2+ entry through the voltage-dependent Ca2+ channels of cells of both these muscles.

Effects of Trimebutine Maleate on Electrical Activities of Isolated Mammalian Cardiac Preparations

The effects of trimebutine maleate on electrical activity in guinea-pig isolated papillary muscles and rabbit sino-atrial nodes have been studied by means of a standard microelectrode method. In papillary muscles, trimebutine (above 10 microM) decreased the maximum rate of rise (Vmax) and the action potential duration at 90% repolarization (APD90), whereas the resting potential was not significantly altered. As to a decrease in Vmax, trimebutine produced a negative shift of the curve relating Vmax to the resting potential along the voltage axis. Trimebutine also depressed the slow action potentials of papillary muscles produced by 27 mM K and 0.2 mM Ba. In spontaneously beating sino-atrial node preparations, trimebutine (above 10 microM) decreased the heart rate, Vmax and the rate of diastolic depolarization. These results indicate that trimebutine maleate possesses a depressant action on the electrical activities of the fast- and slow-response fibres of the heart mainly due to inhibitions of both fast Na+ and slow Ca2+ channels.