Pacemaker activity and ionic currents in mouse atrioventricular node cells

It is well established that Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of I(Na) by tetrodotoxin (TTX) or of I(Ca,L) by isradipine abolished AVNCs pacemaker activity. TTX-resistant (I(Nar)) and TTX-sensitive (I(Nas)) Na(+) currents were recorded in mouse AVNCs, as well as T-(I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents I(Ca,L) density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (I(f)) and that of the fast component of the delayed rectifier current (I(Kr)) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of I(f) by 3 μM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for I(f) in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K(+) current (I(to)). In contrast, no detectable slow component of the delayed rectifier current (I(Ks)) could be recorded in AVNCs. The lower densities of I(f) and I(Ca,L), as well as higher expression of I(Kr) in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.     

Na(+) current contribution to the diastolic depolarization in newborn rabbit SA node cells

Isolated newborn, but not adult, rabbit sinoatrial node (SAN) cells exhibit spontaneous activity that (unlike adult) are highly sensitive to the Na(+) current (I(Na)) blocker TTX. To investigate this TTX action on automaticity, cells were voltage clamped with ramp depolarizations mimicking the pacemaker phase of spontaneous cells (-60 to -20 mV, 35 mV/s). Ramps elicited a TTX-sensitive current in newborn (peak density 0.89 +/- 0.14 pA/pF, n = 24) but not adult (n = 5) cells. When depolarizing ramps were preceded by steplike depolarizations to mimic action potentials, ramp current decreased 54.6 +/- 8.0% (n = 3) but was not abolished. Additional experiments demonstrated that ramp current amplitude depended on the slope of the ramp and that TTX did not alter steady-state holding current at pacemaker potentials. This excluded a steady-state Na(+) window component and suggested a kinetic basis, which was investigated by measuring TTX-sensitive I(Na) during long step depolarizations. I(Na) exhibited a slow but complete inactivation time course at pacemaker voltages (tau = 33.9 +/- 3.9 ms at -50 mV), consistent with the rate-dependent ramp data. The data indicate that owing to slow inactivation of I(Na) at diastolic potentials, a small TTX-sensitive current flows during the diastolic depolarization in neonatal pacemaker myocytes.     

A rapidly activating delayed rectifier K+ current regulates pacemaker activity in adult mouse sinoatrial node cells

We have investigated the physiological role of the "rapidly activating" delayed rectifier K+ current (IKr) in pacemaker activity in isolated sinoatrial node (SAN) myocytes and the expression of mouse ether-a-go-go (mERG) genes in the adult mouse SAN. In isolated, voltage-clamped SAN cells, outward currents evoked by depolarizing steps (greater than -40 mV) were strongly inhibited by the class III methanesulfonanilide compound E-4031 (1-2.5 microM), and the deactivation "tail" currents that occurred during repolarization to a membrane potential of -45 mV were completely blocked. E-4031-sensitive currents (IKr) reached a maximum at a membrane potential of -10 mV and showed pronounced inward rectification at more-positive membrane potentials. Activation of IKr occurred at -40 to 0 mV, with half-activation at about -24 mV. The contribution of IKr to action potential repolarization and diastolic depolarization was estimated by determining the E-4031-sensitive current evoked during voltage clamp with a simulated mouse SAN action potential. IKr reached its peak value (approximately 0.6 pA/pF) near -25 mV, close to the midpoint of the repolarization phase of the simulated action potential, and deactivated almost completely during the diastolic interval. E-4031 (1 microM) slowed the spontaneous pacing rate of Langendorff-perfused, isolated adult mouse hearts by an average of 36.5% (n = 5). Expression of mRNA corresponding to three isoforms coded by the mouse ERG1 gene (mERG1), mERG1a, mERG1a', and mERG1b, was consistently found in the SAN. Our data provide the first detailed characterization of IKr in adult mouse SAN cells, demonstrate that this current plays an important role in pacemaker activity, and indicate that multiple isoforms of mERG1 can contribute to native SAN IKr.     

Characterization of the Participation of Sodium Channels on the Rise in Na+ Induced by 4-Aminopyridine (4-AP) in Synaptosomes

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.     

Role of dual pacemaker mechanisms in sinoatrial node discharge

We investigated whether in the sinoatrial node (SAN) there are two different pacemaker mechanisms and whether either one can maintain spontaneous discharge. These questions were studied by means of an electrophysiological technique and of blockers of different diastolic currents in rabbit and guinea pig isolated SAN. In SAN subsidiary pacemakers of both species, Cs(+) (5-10 mM) or high [K(+)](o) (10-12 mM) decreased the maximum diastolic potential, abolished diastolic depolarization (DD) at polarized levels (subsidiary DD), unmasked a U-shaped dominant DD at depolarized levels, but did not stop the SAN. In rabbit SAN, E4031 (1 microM) and d-sotalol (100 microM) did not stop discharge, but did so after block of subsidiary DD by high [K(+)](o) or Cs(+). In guinea pig SAN, in Tyrode solution E4031, d-sotalol or indapamide (100 microM) did not stop SAN discharge. In the presence of Cs(+) or high [K(+)](o) indapamide (but not E4031 or d-sotalol) stopped the SAN. Ba(2+) (1-5 mM) led to stoppage of discharge both in Tyrode solution and in high [K(+)](o) or Cs(+). Depolarization by blockers of DD unmasked sinusoidal fluctuations, which during recovery were responsible for resumption of discharge. We conclude that in rabbit and guinea pig SAN, two different pacemaker mechanisms (Cs(+)- and K(+)-sensitive subsidiary DD, and Cs(+)- and K(+)-insensitive dominant DD) can independently sustain discharge, but block of both mechanisms leads to quiescence. Abolition of dominant DD by blockers of I(K) is consistent with a decay of I(K) as the dominant pacemaking mechanism, I(Kr) being more important in rabbit and I(Ks) in guinea pig. Sinusoidal fluctuations appear to be an essential component of the pacemaking process.     

Postganglionic nerve stimulation induces temporal inhibition of excitability in rabbit sinoatrial node

Vagal stimulation results in complex changes of pacemaker excitability in the sinoatrial node (SAN). To investigate the vagal effects in the rabbit SAN, we used optical mapping, which is the only technology that allows resolving simultaneous changes in the activation pattern and action potentials morphologies. With the use of immunolabeling, we identified the SAN as a neurofilament 160-positive but connexin 43-negative region (n = 5). Normal excitation originated in the SAN center with a cycle length (CL) of 405 +/- 14 ms (n = 14), spread anisotropically along the crista terminalis (CT), and failed to conduct toward the septum. Postganglionic nerve stimulation (PNS, 400-800 ms) reduced CL by 74 +/- 7% transiently and shifted the leading pacemaker inferiorly (78%) or superiorly (22%) from the SAN center by 2-10 mm. In the intercaval region between the SAN center and the septal block zone, PNS produced an 8 +/- 1-mm(2) region of transient hyperpolarization and inexcitability. The first spontaneous or paced excitation following PNS could not enter this region for 500-1,500 ms. Immunolabeling revealed that the PNS-induced inexcitable region is located between the SAN center and the block zone and has a 2.5-fold higher density of choline acetyltransferase than CT but is threefold lower than the SAN center. The fact that the inexcitability region does not coincide with the most innervated area indicates that the properties of the myocytes themselves, as well as intercellular coupling, must play a role in the inexcitability induction. Optically mapping revealed that PNS resulted in transient loss of pacemaker cell excitability and unidirectional entrance conduction block in the periphery of SAN.     

Altered sinoatrial node function and intra-atrial conduction in murine gain-of-function Scn5a+/ΔKPQ hearts suggest an overlap syndrome

Mutations in SCN5A, the gene encoding the pore-forming subunit of cardiac Na(+) channels, cause a spectrum of arrhythmic syndromes. Of these, sinoatrial node (SAN) dysfunction occurs in patients with both loss- and gain-of-function SCN5A mutations. We explored for corresponding alterations in SAN function and intracardiac conduction and clarified possible mechanisms underlying these in an established mouse long QT syndrome type 3 model carrying a mutation equivalent to human SCN5A-ΔKPQ. Electrophysiological characterizations of SAN function in living animals and in vitro sinoatrial preparations were compared with cellular SAN and two-dimensional tissue models exploring the consequences of Scn5a+/ΔKPQ mutations. Scn5a+/ΔKPQ mice showed prolonged electrocardiographic QT and corrected QT intervals confirming long QT phenotypes. They showed frequent episodes of sinus bradycardia, sinus pause/arrest, and significantly longer sinus node recovery times, suggesting compromised pacemaker activity compared with wild-type mice. Electrocardiographic waveforms suggested depressed intra-atrial, atrioventricular node, and intraventricular conduction in Scn5a+/ΔKPQ mice. Isolated Scn5a+/ΔKPQ sinoatrial preparations similarly showed lower mean intrinsic heart rates and overall slower conduction through the SAN to the surrounding atrium than did wild-type preparations. Computer simulations of both single SAN cells as well as two-dimensional SAN-atrial models could reproduce the experimental observations of impaired pacemaker and sinoatrial conduction in terms of changes produced by both augmented tail and reduced total Na(+) currents, respectively. In conclusion, the gain-of-function long QT syndrome type 3 murine Scn5a+/ΔKPQ cardiac system, in overlap with corresponding features reported in loss-of-function Na(+) channel mutations, shows compromised SAN pacemaker and conduction function explicable in modeling studies through a combination of augmented tail and reduced peak Na(+) currents.     

兔主动脉前庭自律细胞与窦房结电生理特性的比较

为进一步阐明左心室流出道(主动脉前庭)自律细胞的特性,及其与窦房结细胞的异同,本实验利用常规的玻璃微电极细胞内记录技术,观察了一些离子通道阻断剂分别对离体兔窦房结起搏细胞与左心室流出道慢反应自律细胞的电生理特性的影响,重点探讨了这两种自律细胞的0期、4期去极离子流的异同。结果表明:(1)用1μmol/L维拉帕米(verapamil,VER)灌流后,窦房结及主动脉前庭自律细胞的动作电位幅值(APA)、0相最大除极速率(V_(max))、最大舒张电位(MDP)绝对值、舒张期除极速率(VDD)、自发放电频率(RPF)均明显下降,复极90％时间(APD_(90))延长(P<0.05)。(2)用180μmol/L氯化镍(NiCl_2)灌流,两自律细胞的VDD均明显下降;APA、V_(max)和RPF也显著降低,且窦房结细胞的APD_(90)明显延长。(3)给予2 mmol/L 4-氨基吡啶(4-AP)后,窦房结及主动脉前庭自律细胞的VDD均明显增快,MDP绝对值、APA和V_(max)显著下降,APD_(90)明显延长(P<0.05)。(4)给予2 mmol/L氯化铯(CsCl),两自律细胞的VDD及RPF均明显变慢。结果提示:(1)主动脉前庭自发慢反应电位的0相、4相去极离子流及复极离子流均与窦房结优势起搏细胞相似。(2)主动脉前庭起搏细胞Ca~(2+)内流为其0相主要去极离子流,复极过程主要由K~+外流引起,4相自动除极以K~+外流衰减为主,另外     

One-Dimensional Mathematical Model of the Atrioventricular Node Including Atrio-Nodal, Nodal, and Nodal-His Cells

Mathematical models are a repository of knowledge as well as research and teaching tools. Although action potential models have been developed for most regions of the heart, there is no model for the atrioventricular node (AVN). We have developed action potential models for single atrio-nodal, nodal, and nodal-His cells. The models have the same action potential shapes and refractoriness as observed in experiments. Using these models, together with models for the sinoatrial node (SAN) and atrial muscle, we have developed a one-dimensional (1D) multicellular model including the SAN and AVN. The multicellular model has slow and fast pathways into the AVN and using it we have analyzed the rich behavior of the AVN. Under normal conditions, action potentials were initiated in the SAN center and then propagated through the atrium and AVN. The relationship between the AVN conduction time and the timing of a premature stimulus (conduction curve) is consistent with experimental data. After premature stimulation, atrioventricular nodal reentry could occur. After slow pathway ablation or block of the L-type Ca²⁺ current, atrioventricular nodal reentry was abolished. During atrial fibrillation, the AVN limited the number of action potentials transmitted to the ventricle. In the absence of SAN pacemaking, the inferior nodal extension acted as the pacemaker. In conclusion, we have developed what we believe is the first detailed mathematical model of the AVN and it shows the typical physiological and pathophysiological characteristics of the tissue. The model can be used as a tool to analyze the complex structure and behavior of the AVN.     

Role of pacemaking current in cardiac nodes: insights from a comparative study of sinoatrial node and atrioventricular node

Cardiac pacemaking in the sinoatrial (SA) node and atrioventricular (AV) node is generated by an interplay of many ionic currents, one of which is the funny pacemaker current (I_f). To understand the functional role of I_f in two different pacemakers, comparative studies of spontaneous activity and expression of the HCN channel in mouse SA node and AV node were performed. The intrinsic cycle length (CL) is 179±2.7 ms (n=5) in SA node and 258±18.7 ms (n=5) in AV node. Blocking of I_f current by 1 μmol/L ZD7288 increased the CL to 258±18.7 ms (n=5) and 447±92.4 ms (n=5) in SA node and AV node, respectively. However, the major HCN channel, HCN4 expressed at low level in the AV node compared to the SA node. To clarify the discrepancy between the functional importance of I_f and expression level of HCN4 channel, a SA node cell model was used. Increasing the I_f conductance resulted in decreasing in the CL in the model, which explains the high pacemaking rate and high expression of HCN channel in the SA node. Resistance to the blocking of I_f in the SA node might result from compensating effects from other currents (especially voltage sensitive currents) involved in pacemaking. The computer simulation shows that the difference in the intrinsic CL could explain the difference in response to I_f blocking in these two cardiac nodes.     

Slow inactivation of a tetrodotoxin-sensitive current in canine cardiac Purkinje fibers.

We used the two-microelectrode voltage clamp technique and tetrodotoxin (TTX) to investigate the possible occurrence of slow inactivation of sodium channels in canine cardiac Purkinje fibers under physiologic conditions. The increase in net outward current during prolonged (5-20 s) step depolarizations (range -70 to +5 mV) following the application of TTX is time dependent, being maximal immediately following depolarization, and declining thereafter towards a steady value. To eliminate the possibility that this time-dependent current was due to inadequate voltage control of these multicellular preparations early during square clamp pulses, we also used slowly depolarizing voltage clamp ramps (range 5-100 mV/s) to ensure control of membrane potential. TTX-sensitive current also was observed with these voltage ramps; the time dependence of this current was demonstrated by the reduction of the peak current magnitude as the ramp speed was reduced. Reducing the holding potential within the voltage range of sodium channel inactivation also decreased the TTX-sensitive current observed with identical speed ramps. These results suggest that the TTX-sensitive time-dependent current is a direct measure of slow inactivation of canine cardiac sodium channels. This current may play an important role in modulating the action potential duration.     

Freeze-fracture studies of the sinoatrial and atrioventricular nodes of the caprine heart,with special reference to the nexus

Summary The caprine sinoatrial node (SAN) and atrioventricular node (AVN) were studied by freeze-fracture techniques, and their nexus or gap junction structure were compared with that of ordinary atrial and ventricular muscle cells. The general features of the nexus in both the SAN and AVN were essentially identical. Approximately two-thirds of the nexuses observed in the nodal cells consisted of typical macular arrangements of nexal particles, and the remaining third, of atypical configurations of either circular arrangements or linear arrays of particles in continuity with the macular nexuses. Such atypical nexuses were never observed in the ordinary adult myocardial cells. Quantitative analysis revealed that all of the nexuses in the nodal cells measured, were less than 0.1 m², whereas the majority of the nexuses in ordinary myocardial cells (64% in the atrium and 76% in the ventricle) were larger than 0.1 m². No significant differences in diameter and center-to-center distance of nexal particle were found between the nodal cells and ordinary myocardial cells.     

Multiple ryanodine receptor subtypes and heterogeneous ryanodine receptor-gated Ca2+ stores in pulmonary arterial smooth muscle cells

Ryanodine receptors (RyRs) of pulmonary arterial smooth muscle cells (PASMCs) play important roles in major physiological processes such as hypoxic pulmonary vasoconstriction and perinatal pulmonary vasodilatation. Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca2+ stores are distributed heterogeneously in systemic vascular myocytes. However, the molecular identity and subcellular distribution of RyRs have not been examined in PASMCs. In this study we detected mRNA and proteins of all three subtypes in rat intralobar PASMCs using RT-PCR and Western blot. Quantitative real-time RT-PCR showed that RyR2 mRNA was most abundant, approximately 15-20 times more than the other two subtypes. Confocal fluorescence microscopy revealed that RyRs labeled with BODIPY TR-X ryanodine were localized in the peripheral and perinuclear regions and were colocalized with sarcoplasmic reticulum labeled with Fluo-5N. Immunostaining showed that the subsarcolemmal regions exhibited clear signals of RyR1 and RyR2, whereas the perinuclear compartments contained mainly RyR1 and RyR3. Ca2+ sparks were recorded in both regions, and their activities were enhanced by a subthreshold concentration of caffeine or by endothelin-1, indicating functional RyR-gated Ca2+ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) = 193.3 +/- 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca2+ sparks (FDHM = 44.6 +/- 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca2+ stores; the molecular complexity and spatial heterogeneity of RyRs may facilitate specific Ca2+ regulation of cellular functions in PASMCs.     

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.     

Temperature acclimation modifies sinoatrial pacemaker mechanism of the rainbow trout heart

The hypothesis of pacemaker level origin of thermal compensation in heart rate was tested by recording action potentials (AP) in intact sinoatrial tissue and enzymatically isolated pacemaker cells of rainbow trout acclimated at 4 degrees C (cold) and 18 degrees C (warm). With electrophysiological recordings, the primary pacemaker was located at the base of the sinoatrial valve, where a morphologically distinct ring of tissue comprising myocytes and neural elements was found by histological examination. Intrinsic beating rate of this pacemaker was higher in cold-acclimated (46 +/- 6 APs/min) than warm-acclimated trout (38 +/- 3 APs/min; P < 0.05), and a similar difference was seen in beating rate of isolated pacemaker cells (44 +/- 6 vs. 38 +/- 6 APs/min; P < 0.05), supporting the hypothesis that thermal acclimation modifies the intrinsic pacemaker mechanism of fish heart. Inhibition of sarcoplasmic reticulum (SR) with 10 microM ryanodine and 1 microM thapsigargin did not affect heart rate in either warm- or cold-acclimated trout at 11 degrees C but reduced heart rate in warm-acclimated trout from 74 +/- 2 to 42 +/- 6 APs/min (P < 0.05) at 18 degrees C. At 11 degrees C, a half-maximal blockade of the delayed rectifier K+ current (I(Kr)) with 0.1 microM E-4031 reduced heart rate more in warm-acclimated (from 45 +/- 1 to 24 +/- 5 APs/min) than cold-acclimated trout (56 +/- 3 vs. 48 +/- 2 APs/min), whereas I(Kr) density was higher and AP duration less in cold-acclimated trout (P > 0.05). Collectively, these findings suggest that a cold-induced increase in AP discharge frequency is at least partly due to higher density of the I(Kr) in the cold-acclimated trout, whereas contribution of SR Ca2+ release to thermal compensation of heart rate is negligible.     

Mouse models for studying pacemaker channel function and sinus node arrhythmia

Pacemaker activity of the heart is generated by a small group of cells forming the sinoatrial node (SAN). Cells of the SAN are spontaneously active and generate action potentials with remarkable regularity and stability under all physiological conditions. The exact molecular mechanisms underlying pacemaker potentials in the SAN have not yet been fully elucidated. Several voltage-dependent ion channels as well as intracellular calcium cycling processes are thought to contribute to the pacemaker activity. Hyperpolarization-activated cation channels, which generate the I_f current, have biophysical properties which seem ideally suited for the initiation of spontaneous electrical activity. This review describes recent work on several transgenic mice lacking different cardiac HCN channel subtypes. The role of I_f for normal pacemaking and sinus node arrhythmia as revealed by these genetic models will be discussed. In addition, a new mouse line is described which enables gene targeting in a temporally-controlled manner selectively in SAN cells. Elucidating the function of HCN and other ion channels in well-controlled mouse models should ultimately lead to a better understanding of the mechanisms underlying human sinoatrial arrhythmias.     

Four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes

In the experiments here, the time- and voltage-dependent properties of the Ca2+-independent, depolarization-activated K+ currents in adult mouse ventricular myocytes were characterized in detail. In the majority (65 of 72, approximately 90%) of cells dispersed from the ventricles, analysis of the decay phases of the outward currents revealed three distinct K+ current components: a rapidly inactivating, transient outward K+ current, Ito,f (mean +/- SEM taudecay = 85 +/- 2 ms); a slowly (mean +/- SEM taudecay = 1,162 +/- 29 ms) inactivating K+ current, IK,slow; and a non inactivating, steady state current, Iss. In a small subset (7 of 72, approximately 10%) of cells, Ito,f was absent and a slowly inactivating (mean +/- SEM taudecay = 196 +/- 7 ms) transient outward current, referred to as Ito,s, was identified; the densities and properties of IK,slow and Iss in Ito,s-expressing cells are indistinguishable from the corresponding currents in cells with Ito,f. Microdissection techniques were used to remove tissue pieces from the left ventricular apex and from the ventricular septum to allow the hypothesis that there are regional differences in Ito,f and Ito,s expression to be tested directly. Electrophysiological recordings revealed that all cells isolated from the apex express Ito,f (n = 35); Ito,s is not detected in these cells (n = 35). In the septum, by contrast, all of the cells express Ito,s (n = 28) and in the majority (22 of 28, 80%) of cells, Ito,f is also present. The density of Ito,f (mean +/- SEM at +40 mV = 6.8 +/- 0.5 pA/pF, n = 22) in septum cells, however, is significantly (P < 0.001) lower than Ito,f density in cells from the apex (mean +/- SEM at +40 mV = 34.6 +/- 2.6 pA/pF, n = 35). In addition to differences in inactivation kinetics, Ito,f, Ito,s, and IK,slow display distinct rates of recovery (from inactivation), as well as differential sensitivities to 4-aminopyridine (4-AP), tetraethylammonium (TEA), and Heteropoda toxin-3. IK,slow, for example, is blocked selectively by low (10-50 microM) concentrations of 4-AP and by (>/=25 mM) TEA. Although both Ito,f and Ito,s are blocked by high (>100 microM) 4-AP concentrations and are relatively insensitive to TEA, Ito,f is selectively blocked by nanomolar concentrations of Heteropoda toxin-3, and Ito,s (as well as IK,slow and Iss) is unaffected. Iss is partially blocked by high concentrations of 4-AP or TEA. The functional implications of the distinct properties and expression patterns of Ito,f and Ito,s, as well as the likely molecular correlates of these (and the IK,slow and Iss) currents, are discussed.     

Characterization of Na(+) currents in isolated dorsal unpaired median neurons of Locusta migratoria and effect of the alpha-like scorpion toxin BmK M1

A primary cell culture was developed for efferent dorsal unpaired median (DUM) neurons of the locust. The isolated somata were able to generate Tetrodotoxin (TTX)-sensitive action potentials in vitro. The alpha-like scorpion toxin BmK M1, from the Asian scorpion Buthus martensi Karsch, prolonged the duration of the action potential up to 50 times. To investigate the mechanism of action of BmK M1, the TTX-sensitive voltage gated Na(+) currents were studied in detail using the whole cell patch clamp technique. BmK M1 slowed down and partially inhibited the inactivation of the TTX-sensitive Na(+) current in a dose dependent manner (EC50=326.8+/-34.5 nM). Voltage and time dependence of the Na(+) current were described in terms of the Hodgkin-Huxley model and compared in control conditions and in the presence of 500 nM BmK M1. The BmK M1 shifted steady state inactivation by 10.8 mV to less negative potentials. The steady state activation was shifted by 5.5 mV to more negative potentials, making the activation window larger. Moreover, BmK M1 increased the fast time constant of inactivation, leaving the activation time constant unchanged. In summary, BmK M1 primarily affected the inactivation parameters of the voltage gated Na(+) current in isolated locust DUM neurons.     

Electrophysiological characteristics of cardiac pacemaker cells of the frog Caudiverbera caudiverbera

1. The cardiac pacemaker cells of the frog Caudiverbera caudiverbera are centrally located in the sinus venosus. These cells are rounded, smaller than contractile fibres and have large nuclei. 2. Intracellular recording confirmed the existence of primary and transitional pacemaker cells. 3. Action potentials from primary cells were resistant to blockade by tetrodotoxin (TTX), but were abolished by verapamil suggesting that their bioelectric activity is dependent on a slow inward current. 4. Transitional cells appeared to have two different inward currents contributing to the upstroke: a fast TTX-sensitive and a slow verapamil-sensitive current.