Modulation of action potential by [Ca2+]i in modeled rat atrial and guinea pig ventricular myocytes

We simulated mechanisms that increase Ca2+ transients with two models: the Luo-Rudy II model for guinea pig (GP) ventricle (GP model) representing long action potential (AP) myocytes and the rat atrial (RA) model exemplifying myocytes with short APs. The interventions were activation of stretch-gated cationic channels, increase of intracellular Na+ concentration ([Na+]i), simulated bet-adrenoceptor stimulation, and Ca2+ accumulation into the sarcoplasmic reticulum (SR). In the RA model, interventions caused an increase of AP duration. In the GP model, AP duration decreased except in the simulated beta-stimulation where it lengthened APs as in the RA model. We conclude that the changes in the APs are significantly contributed by the increase of the Ca2+ transient itself. The AP duration is controlled differently in cardiac myocytes with short and long AP durations. With short APs, an increase of the Ca2+ transient promotes an inward current via Na+/Ca2+-exchanger lengthening the AP. This effect is similar regardless of the mechanism causing the increase of the Ca2+ transient. With long APs the Ca2+ transient increase decreases the AP duration via inactivation of the L-type Ca2+ current. However, L-type current increase (as with beta-stimulation) increases the AP duration despite the simultaneous Ca2+ transient augmentation. The results explain the dispersion of AP changes in myocytes with short and long APs during interventions increasing the Ca2+ transients.     

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.     

In silico assessment of Y1795C and Y1795H SCN5A mutations: implication for inherited arrhythmogenic syndromes

The effects of two SCN5A mutations (Y1795C, Y1795H), previously identified in one Long QT syndrome type 3 (LQT3) and one Brugada syndrome (BrS) families, were investigated by means of numerical modeling of ventricular action potential (AP). A Markov model capable of reproducing a wild-type as well as a mutant sodium current (I(Na)) was identified and was included into the Luo-Rudy ventricular cell model for action potential (AP) simulation. The characteristics of endocardial, midmyocardial, and epicardial cells were reproduced by differentiating the transient outward current (I(TO)) and the ratio of slow delayed rectifier potassium (I(Ks)) to rapid delayed rectifier current (I(Kr)). Administration of flecainide and mexiletine was simulated by appropriately modifying I(Na), calcium current (I(Ca)), I(TO), and I(Kr). Y1795C prolonged AP in a rate-dependent manner, and early afterdepolarizations (EADs) appeared during bradycardia in epicardial and midmyocardial cells; flecainide and mexiletine shortened AP and abolished EADs. Y1795H resulted in minimal changes in the APs; flecainide but not mexiletine induced APs heterogeneity across the ventricular wall that accounts for the ST segment elevation induced by flecainide in Y1795H carriers. The AP abnormalities induced by Y1795H and Y1795C can explain the clinically observed surface ECG phenotype. For the first time by modeling the effects of flecainide and mexiletine, we are able to gather mechanistic insights on the response to drugs administration observed in affected patients.     

Biophysical properties and slow voltage-dependent inactivation of a sustained sodium current in entorhinal cortex layer-II principal neurons: a whole-cell and single-channel study.

The functional and biophysical properties of a sustained, or "persistent," Na(+) current (I(NaP)) responsible for the generation of subthreshold oscillatory activity in entorhinal cortex layer-II principal neurons (the "stellate cells") were investigated with whole-cell, patch-clamp experiments. Both acutely dissociated cells and slices derived from adult rat entorhinal cortex were used. I(NaP), activated by either slow voltage ramps or long-lasting depolarizing pulses, was prominent in both isolated and, especially, in situ neurons. The analysis of the gating properties of the transient Na(+) current (I(NaT)) in the same neurons revealed that the resulting time-independent "window" current (I(NaTW)) had both amplitude and voltage dependence not compatible with those of the observed I(NaP), thus implying the existence of an alternative mechanism of persistent Na(+)-current generation. The tetrodotoxin-sensitive Na(+) currents evoked by slow voltage ramps decreased in amplitude with decreasing ramp slopes, thus suggesting that a time-dependent inactivation was taking place during ramp depolarizations. When ramps were preceded by increasingly positive, long-lasting voltage prepulses, I(NaP) was progressively, and eventually completely, inactivated. The V(1/2) of I(NaP) steady state inactivation was approximately -49 mV. The time dependence of the development of the inactivation was also studied by varying the duration of the inactivating prepulse: time constants ranging from approximately 6.8 to approximately 2.6 s, depending on the voltage level, were revealed. Moreover, the activation and inactivation properties of I(NaP) were such as to generate, within a relatively broad membrane-voltage range, a really persistent window current (I(NaPW)). Significantly, I(NaPW) was maximal at about the same voltage level at which subthreshold oscillations are expressed by the stellate cells. Indeed, at -50 mV, the I(NaPW) was shown to contribute to >80% of the persistent Na(+) current that sustains the subthreshold oscillations, whereas only the remaining part can be attributed to a classical Hodgkin-Huxley I(NaTW). Finally, the single-channel bases of I(NaP) slow inactivation and I(NaPW) generation were investigated in cell-attached experiments. Both phenomena were found to be underlain by repetitive, relatively prolonged late channel openings that appeared to undergo inactivation in a nearly irreversible manner at high depolarization levels (-10 mV), but not at more negative potentials (-40 mV).     

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: Ca(2+) versus voltage dependent inactivation of L-type Ca(2+) current (I(CaL)); kinetics for the transient outward, rapid delayed rectifier (I(Kr)), Na(+)/Ca(2+) exchange (I(NaCa)), 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 Ca(2+) (including peak and decay) and intracellular sodium ([Na(+)](i)) in undiseased human myocytes were quantitatively reproduced by the model. Early afterdepolarizations were induced by I(Kr) block during slow pacing, and AP and Ca(2+) alternans appeared at rates >200 bpm, as observed in the nonfailing human ventricle. Ca(2+)/calmodulin-dependent protein kinase II (CaMK) modulated rate dependence of Ca(2+) cycling. I(NaCa) linked Ca(2+) 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 I(CaL) were also contributors. APD shortening during restitution was primarily dependent on reduced late Na(+) and I(CaL) currents due to inactivation at short diastolic intervals, with additional contribution from elevated I(Kr) due to incomplete deactivation.     

Contribution of slowly inactivating potassium current to delayed firing of action potentials in NG108-15 neuronal cells: experimental and theoretical studies

The properties of slowly inactivating delayed-rectifier K⁺ current (I_Kdr) were investigated in NG108-15 neuronal cells differentiated with long-term exposure to dibutyryl cyclic AMP. Slowly inactivating I_Kdr could be elicited by prolonged depolarizations from −50 to +50 mV. These outward K⁺ currents were found to decay at potentials above −20 mV, and the decay became faster with greater depolarization. Cell exposure to aconitine resulted in the reduction of I_Kdr amplitude along with an accelerated decay of current inactivation. Under current-clamp recordings, a delay in the initiation of action potentials (APs) in response to prolonged current stimuli was observed in these cells. Application of aconitine shortened the AP initiation in combination with an increase in both width of spike discharge and firing frequency. The computer model, in which state-dependent inactivation of I_Kdr was incorporated, was also implemented to predict the firing behavior present in NG108-15 cells. As the inactivation rate constant of I_Kdr was elevated, the firing frequency was progressively increased along with a shortening of the latency for AP appearance. Our theoretical work and the experimental results led us to propose a pivotal role of slowly inactivating I_Kdr in delayed firing of APs in NG108-15 cells. The results also suggest that aconitine modulation of I_Kdr gating is an important molecular mechanism through which it can contribute to neuronal firing.     

Quantitative comparison of cardiac ventricular myocyte electrophysiology and response to drugs in human and nonhuman species

Explanations for arrhythmia mechanisms at the cellular level are usually based on experiments in nonhuman myocytes. However, subtle electrophysiological differences between species may lead to different rhythmic or arrhythmic cellular behaviors and drug response given the nonlinear and highly interactive cellular system. Using detailed and quantitatively accurate mathematical models for human, dog, and guinea pig ventricular action potentials (APs), we simulated and compared cell electrophysiology mechanisms and response to drugs. Under basal conditions (absence of β-adrenergic stimulation), Na(+)/K(+)-ATPase changes secondary to Na(+) accumulation determined AP rate dependence for human and dog but not for guinea pig where slow delayed rectifier current (I(Ks)) was the major rate-dependent current. AP prolongation with reduction of rapid delayed rectifier current (I(Kr)) and I(Ks) (due to mutations or drugs) showed strong species dependence in simulations, as in experiments. For humans, AP prolongation was 80% following I(Kr) block. It was 30% for dog and 20% for guinea pig. Under basal conditions, I(Ks) block was of no consequence for human and dog, but for guinea pig, AP prolongation after I(Ks) block was severe. However, with β-adrenergic stimulation, I(Ks) played an important role in all species, particularly in AP shortening at fast rate. Quantitative comparison of AP repolarization, rate-dependence mechanisms, and drug response in human, dog, and guinea pig revealed major species differences (e.g., susceptibility to arrhythmogenic early afterdepolarizations). Extrapolation from animal to human electrophysiology and drug response requires great caution.     

Spontaneous action potential generation due to persistent sodium channel currents in simulated carotid body afferent fibers.

The mechanism by which action potentials (APs) are generated in afferent nerve fibers in the carotid body is unknown, but it is generally speculated to be release of an excitatory transmitter and synaptic depolarizing events. However, previous results suggested that Na(+) channels in the afferent nerve fibers play an important role in this process. To better understand the potential mechanism by which Na(+) channels may generate APs, a mathematical model of chemoreceptor nerve fibers that incorporated Hodgkin-Huxley-type Na(+) channels with kinetics of activation and inactivation, as determined previously from recordings of petrosal chemoreceptor neurons, was constructed. While the density of Na(+) channels was kept constant, spontaneous APs arose in nerve terminals as the axonal diameter was reduced to that in rat carotid body. AP excitability and pattern were similar to those observed in chemoreceptor recordings: 1) a random pattern at low- and high-frequency discharge rates, 2) a high sensitivity to reductions in extracellular Na(+) concentration, and 3) a variation in excitability that increased with AP generation rate. Taken together, the results suggest that an endogenous process in chemoreceptor nerve terminals may underlie AP generation, a process independent of synaptic depolarizing events.     

In silico study of action potential and QT interval shortening due to loss of inactivation of the cardiac rapid delayed rectifier potassium current

The rapid delayed rectifier K(+) current, I(Kr), plays a key role in repolarisation of cardiac ventricular action potentials (APs). In recent years, a novel clinical condition denoted the short QT syndrome (SQTS) has been identified and, very recently, gain in function mutations in the gene encoding the pore-forming sub-unit of the I(Kr) channel have been proposed to underlie SQTS in some patients. Here, computer simulations were used to investigate the effects of the selective loss of voltage-dependent inactivation of I(Kr) upon ventricular APs and on the QT interval of the electrocardiogram. I(Kr) and inactivation-deficient I(Kr) were incorporated into Luo-Rudy ventricular AP models. Inactivation-deficient I(Kr) produced AP shortening that was heterogeneous between endocardial, mid-myocardial, and epicardial ventricular cell models, irrespective of whether heterogeneity between these sub-regions was incorporated of slow delayed rectifier K(+) current (I(Ks)) alone, or of I(Ks) together with that of transient outward K(+) current. The selective loss of rectification of I(Kr) did not augment transmural dispersion of AP repolarisation, as AP shortening was greater in mid-myocardial than in endo- or epicardial cell models. Simulated conduction through a 1 D transmural ventricular strand was altered by incorporation of inactivation-deficient I(Kr) and the reconstructed QT interval was shortened. Collectively, these results substantiate the notion that selective loss of I(Kr) inactivation produces a gain in I(Kr) function that causes QT interval shortening.     

Roles of I(f) and intracellular Ca2+ release in spontaneous activity of ventricular cardiomyocytes during murine embryonic development

In recent years, the contribution of I(f), an important pacemaker current, and intracellular Ca²⁺ release (ICR) from sarcoplasmic reticulum to pacemaking and arrhythmia has been intensively studied. However, their functional roles in embryonic heart remain uncertain. Using patch clamp, Ca²⁺ imaging, and RT‐PCR, we found that I(f) regulated the firing rate in early and late stage embryonic ventricular cells, as ivabradine (30 µM), a specific blocker of I(f), slowed down action potential (AP) frequency. This inhibitory effect was even stronger in late stage cells, though I(f) was down‐regulated. In contrast to I(f), ICR was found to be indispensable for the occurrence of APs in ventricular cells of different stages, because abolishment of ICR with ryanodine and 2‐aminoethoxydiphenyl borate (2‐APB), specific blockers of ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs), completely abolished APs. In addition, we noticed that RyR‐ and IP3R‐mediated ICR coexisted in early‐stage ventricular cells and RyRs functionally dominated. While at late stage RyRs, but not IP3Rs, mediated ICR. In both early and late stage ventricular cells, Na‐Ca exchanger current (I_Na/Ca) mediated ICR‐triggered depolarization of membrane potential and resulted in the initiation of APs. We also observed that different from I(f), which presented as the substantial component of the earlier diastolic depolarization current, application of ryanodine, and/or 2‐APB slowed the late phase of diastolic depolarization. Thus, we conclude that in murine embryonic ventricular cells I(f) regulates firing rate, while RyRs and IP3Rs (early stage) or RyRs (late stage)‐mediated ICR determines the occurrence of APs. J. Cell. Biochem. 114: 1852–1862, 2013. © 2013 Wiley Periodicals, Inc.     

Amplitude-dependent spike-broadening and enhanced Ca(2+) signaling in GnRH-secreting neurons

In GnRH-secreting (GT1) neurons, activation of Ca(2+)-mobilizing receptors induces a sustained membrane depolarization that shifts the profile of the action potential (AP) waveform from sharp, high-amplitude to broad, low-amplitude spikes. Here we characterize this shift in the firing pattern and its impact on Ca(2+) influx experimentally by using prerecorded sharp and broad APs as the voltage-clamp command pulse. As a quantitative test of the experimental data, a mathematical model based on the membrane and ionic current properties of GT1 neurons was also used. Both experimental and modeling results indicated that inactivation of the tetrodotoxin-sensitive Na(+) channels by sustained depolarization accounted for a reduction in the amplitude of the spike upstroke. The ensuing decrease in tetraethylammonium-sensitive K(+) current activation slowed membrane repolarization, leading to AP broadening. This change in firing pattern increased the total L-type Ca(2+) current and facilitated AP-driven Ca(2+) entry. The leftward shift in the current-voltage relation of the L-type Ca(2+) channels expressed in GT1 cells allowed the depolarization-induced AP broadening to facilitate Ca(2+) entry despite a decrease in spike amplitude. Thus the gating properties of the L-type Ca(2+) channels expressed in GT1 neurons are suitable for promoting AP-driven Ca(2+) influx in receptor- and non-receptor-depolarized cells.     

A mathematical model of action potential heterogeneity in adult rat left ventricular myocytes.

Mathematical models were developed to reconstruct the action potentials (AP) recorded in epicardial and endocardial myocytes isolated from the adult rat left ventricle. The main goal was to obtain additional insight into the ionic mechanisms responsible for the transmural AP heterogeneity. The simulation results support the hypothesis that the smaller density and the slower reactivation kinetics of the Ca(2+)-independent transient outward K(+) current (I(t)) in the endocardial myocytes can account for the longer action potential duration (APD), and more prominent rate dependence in that cell type. The larger density of the Na(+) current (I(Na)) in the endocardial myocytes results in a faster upstroke (dV/dt(max)). This, in addition to the smaller magnitude of I(t), is responsible for the larger peak overshoot of the simulated endocardial AP. The prolonged APD in the endocardial cell also leads to an enhanced amplitude of the sustained K(+) current (I(ss)), and a larger influx of Ca(2+) ions via the L-type Ca(2+) current (I(CaL)). The latter results in an increased sarcoplasmic reticulum (SR) load, which is mainly responsible for the higher peak systolic value of the Ca(2+) transient [Ca(2+)](i), and the resultant increase in the Na(+)-Ca(2+) exchanger (I(NaCa)) activity, associated with the simulated endocardial AP. In combination, these calculations provide novel, quantitative insights into the repolarization process and its naturally occurring transmural variations in the rat left ventricle.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

First node of Ranvier facilitates high-frequency burst encoding

In central neurons the first node of Ranvier is located at the first axonal branchpoint, ～ 100 μm from the axon initial segment where synaptic inputs are integrated and converted into action potentials (APs). Whether the first node contributes to this signal transformation is not well understood. Here it was found that in neocortical layer 5 axons, the first branchpoint is required for intrinsic high-frequency (≥ 100 Hz) AP bursts. Furthermore, block of nodal Na(+) channels or axotomy of the first node in intrinsically bursting neurons depolarized the somatic AP voltage threshold (～ 5 mV) and eliminated APs selectively within a high-frequency cluster in response to steady currents or simulated synaptic inputs. These results indicate that nodal persistent Na(+) current exerts an anterograde influence on AP initiation in the axon initial segment, revealing a computational role of the first node of Ranvier beyond conduction of the propagating AP.     

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.     

Differential Regulation of Action Potentials by Inactivating and Noninactivating BK Channels in Rat Adrenal Chromaffin Cells

Large-conductance Ca²⁺-activated K⁺ (BK) channels can regulate cellular excitability in complex ways because they are able to respond independently to two distinct cellular signals, cytosolic Ca²⁺ and membrane potential. In rat chromaffin cells (RCC), inactivating BK_i and noninactivating (BK_s) channels differentially contribute to RCC action potential (AP) firing behavior. However, the basis for these differential effects has not been fully established. Here, we have simulated RCC action potential behavior, using Markovian models of BK_i and BK_s current and other RCC currents. The analysis shows that BK current influences both fast hyperpolarization and afterhyperpolarization of single APs and that, consistent with experimental observations, BK_i current facilitates repetitive firing of APs, whereas BK_s current does not. However, the key functional difference between BK_i and BK_s current that accounts for the differential firing is not inactivation but the more negatively shifted activation range for BK_i current at a given [Ca²⁺].     

Reactivation processes of three inward current systems involved in the rising phase of the action potentials in embryonic chick hearts

In embryonic chick hearts during development, there are three inward current systems which are involved in the rising phases of the action potentials (APs): fast INa, slow ICa, and tetrodotoxin-insensitive slow INa. To assess reactivation processes for these three types of inward current channels (fast Na+, slow Ca2+, and slow Na+ channels), diastolic recovery of Vmax was examined in embryonic chick hearts using a paired-pulse protocol. In all cases, the diastolic recoveries were approximated by single exponential functions. The time constants of recovery (tau(V)) and T90% (the diastolic interval which allows 90% recovery of Vmax of the premature AP) were, respectively, 53.1 +/- 5.2 and 61.5 +/- 8.6 ms for Na+-dependent fast AP (n = 10), 376.9 +/- 49.3 and 659.2 +/- 113.1 ms for the Ca2+-dependent slow AP (n = 10), and 40.7 +/- 5.3 and 45.6 +/- 12.0 ms for the Na+-dependent slow AP (n = 10). In the presence of lidocaine, the recovery kinetics also appeared to be single exponentials for diastolic intervals up to 500 ms (fast APs) or 250 ms (slow APs). The reactivation processes for the Na+-dependent fast and slow channels were significantly slowed by 100 microM lidocaine. In addition, in the presence of 100 microM lidocaine, Vmax was depressed in a frequency-dependent manner; the higher the stimulation frequency, the greater the depression. Hence, the fast Na+ channels and the slow Na+ channels had the following similarities: rapid reactivation, reactivation slowed by lidocaine, and frequency-dependent depression in the presence of lidocaine.     

Mytilus inhibitory peptide (MIP) induces a Na+-activated K+-current in snail neurons

Two microelectrode voltage-clamp and single-channel recordings were performed on D-cluster neurons of snail right parietal ganglion in order to study the properties of MIP-activated potassium current. It was found that the octapeptide member of the MIP-family, ASHIPRFVa elicits an outward current, which possesses all the properties characteristic for the hexapeptide(s) inward membrane response. The main component of the peptide elicited response is highly [K+]o dependent, however the response was attenuated in Na-free extracellular saline. The peptide elicited response was mimicked by raising the [Na+]i by pressure injection of Na+ into the cell. Single channel recordings indicated that MIP-induced outward K-current is Na-dependent. The probability to find a channel in open state increases with increasing intracellular Na+-concentration. Excised inside-out patches obtained from D-neurons contained I(K(Na)) channels could be activated by exposure of the cytoplasmic face of the patch membrane to 40 mM Na+, and 40 mM Li+, as well. The single channel current amplitude at -60 mV is 15 pA and the single channel conductance is 212 pS between -80 and 0 mV. It was concluded that MIP's activate a novel type of K+-current in the snail neurons. This current is the Na-activated K+-current. The single channel properties of the MIP activated channel is in concert with I(K(Na)) data obtained on different vertebrate and invertebrate preparations.