Impaired Inactivation of L-Type Ca2+ Current as a Potential Mechanism for Variable Arrhythmogenic Liability of HERG K+ Channel Blocking Drugs

The proarrhythmic effects of new drugs have been assessed by measuring rapidly activating delayed-rectifier K⁺ current (I_Kr) antagonist potency. However, recent data suggest that even drugs thought to be highly specific I_Kr blockers can be arrhythmogenic via a separate, time-dependent pathway such as late Na⁺ current augmentation. Here, we report a mechanism for a quinolone antibiotic, sparfloxacin-induced action potential duration (APD) prolongation that involves increase in late L-type Ca²⁺ current (I_CaL) caused by a decrease in Ca²⁺-dependent inactivation (CDI). Acute exposure to sparfloxacin, an I_Kr blocker with prolongation of QT interval and torsades de pointes (TdP) produced a significant APD prolongation in rat ventricular myocytes, which lack I_Kr due to E4031 pretreatment. Sparfloxacin reduced peak I_CaL but increased late I_CaL by slowing its inactivation. In contrast, ketoconazole, an I_Kr blocker without prolongation of QT interval and TdP produced reduction of both peak and late I_CaL, suggesting the role of increased late I_CaL in arrhythmogenic effect. Further analysis showed that sparfloxacin reduced CDI. Consistently, replacement of extracellular Ca²⁺ with Ba²⁺ abolished the sparfloxacin effects on I_CaL. In addition, sparfloxacin modulated I_CaL in a use-dependent manner. Cardiomyocytes from adult mouse, which is lack of native I_Kr, demonstrated similar increase in late I_CaL and afterdepolarizations. The present findings show that sparfloxacin can prolong APD by augmenting late I_CaL. Thus, drugs that cause delayed I_CaL inactivation and I_Kr blockage may have more adverse effects than those that selectively block I_Kr. This mechanism may explain the reason for discrepancies between clinically reported proarrhythmic effects and I_Kr antagonist potencies.     

Simulation of Ca-calmodulin-dependent protein kinase II on rabbit ventricular myocyte ion currents and action potentials

Ca-calmodulin-dependent protein kinase II (CaMKII) was recently shown to alter Na⁺ channel gating and recapitulate a human Na⁺ channel genetic mutation that causes an unusual combined arrhythmogenic phenotype in patients: simultaneous long QT syndrome and Brugada syndrome. CaMKII is upregulated in heart failure where arrhythmias are common, and CaMKII inhibition can reduce arrhythmias. Thus, CaMKII-dependent channel modulation may contribute to acquired arrhythmic disease. We developed a Markovian Na⁺ channel model including CaMKII-dependent changes, and incorporated it into a comprehensive myocyte action potential (AP) model with Na⁺ and Ca²⁺ transport. CaMKII shifts Na⁺ current (I_Na) availability to more negative voltage, enhances intermediate inactivation, and slows recovery from inactivation (all loss-of-function effects), but also enhances late noninactivating I_Na (gain of function). At slow heart rates, with long diastolic time for I_Na recovery, late I_Na is the predominant effect, leading to AP prolongation (long QT syndrome). At fast heart rates, where recovery time is limited and APs are shorter, there is little effect on AP duration, but reduced availability decreases I_Na, AP upstroke velocity, and conduction (Brugada syndrome). CaMKII also increases cardiac Ca²⁺ and K⁺ currents (I_Ca and I_to), complicating CaMKII-dependent AP changes. Incorporating I_Ca and I_to effects individually prolongs and shortens AP duration. Combining I_Na, I_Ca, and I_to effects results in shortening of AP duration with CaMKII. With transmural heterogeneity of I_to and I_to downregulation in heart failure, CaMKII may accentuate dispersion of repolarization. This provides a useful initial framework to consider pathways by which CaMKII may contribute to arrhythmogenesis.     

Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca(2+) current in cardiomyocytes

Oxidative stress remodels Ca²⁺ signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca²⁺ signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca²⁺ current (I_Ca,L) in rat cardiomyocytes. A 5-min exposure of 1 mM H₂O₂ induced an increase in I_Ca,L, and this increase was sustained for ~ 1 h. The CaMKII inhibitor KN-93 fully reversed H₂O₂-induced LTF of I_Ca,L, indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca²⁺ release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H₂O₂ via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.     

A Mathematical Model of the Electrophysiological Alterations in Rat Ventricular Myocytes in Type-I Diabetes

Our mathematical model of the rat ventricular myocyte (Pandit et al., 2001) was utilized to explore the ionic mechanism(s) that underlie the altered electrophysiological characteristics associated with the short-term model of streptozotocin-induced, type-I diabetes. The simulations show that the observed reductions in the Ca²⁺-independent transient outward K⁺ current (I_t) and the steady-state outward K⁺ current (I_ss), along with slowed inactivation of the L-type Ca²⁺ current (I_CaL), can result in the prolongation of the action potential duration, a well-known experimental finding. In addition, the model demonstrates that the slowed reactivation kinetics of I_t in diabetic myocytes can account for the more pronounced rate-dependent action potential duration prolongation in diabetes, and that a decrease in the electrogenic Na⁺-K⁺ pump current (I_NaK) results in a small depolarization in the resting membrane potential (V_rest). This depolarization reduces the availability of the Na⁺ channels (I_Na), thereby resulting in a slower upstroke (dV/dt_max) of the diabetic action potential. Additional simulations suggest that a reduction in the magnitude of I_CaL, in combination with impaired sarcoplasmic reticulum uptake can lead to a decreased sarcoplasmic reticulum Ca²⁺ load. These factors contribute to characteristic abnormal [Ca²⁺]_i homeostasis (reduced peak systolic value and rate of decay) in myocytes from diabetic animals. In combination, these simulation results provide novel information and integrative insights concerning plausible ionic mechanisms for the observed changes in cardiac repolarization and excitation-contraction coupling in rat ventricular myocytes in the setting of streptozotocin-induced, type-I diabetes.     

Mechanisms of a Human Skeletal Myotonia Produced by Mutation in the C-Terminus of NaV1.4: Is Ca2+ Regulation Defective?

Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNa_V1.4_F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNa_V1.4_F1705I are incompletely understood. The location of the hNa_V1.4_F1705I in the CT prompted us to examine the role of Ca²⁺ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human Na_V1.4 channel regulation by Ca²⁺ and CaM. hNa_V1.4_F1705I inactivation gating is Ca²⁺-sensitive compared to wild type hNa_V1.4 which is Ca²⁺ insensitive and the mutant channel exhibits a depolarizing shift of the V_1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNa_V1.4 channel background (rNa_V1.4_F1698I) eliminates Ca²⁺ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca²⁺ sensitivity of gating between wild type and mutant human and rat Na_V1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca²⁺/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows I_Na decay in a Ca²⁺-sensitive fashion. The combination of the altered voltage dependence and kinetics of I_Na decay contribute to the myotonic phenotype and may involve the Ca²⁺-sensing apparatus in the CT of Na_V1.4.     

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca²⁺ current through both enhancement of Ca²⁺ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na⁺–H⁺ exchange (NHE), we examined the possible role of NHE in the regulation of I_Ca during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm²) fluid was applied onto single rat ventricular myocytes, and the I_Ca was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the I_Ca (IC₅₀ = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the I_Ca in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the I_Ca under FP was not altered by intracellular high Ca²⁺ buffering. Replacement of external Cl⁻ with aspartate to inhibit the Cl⁻-dependent acid loader eliminated the inhibitory effect of HOE642 on I_Ca. These results suggest that NHE may attenuate FP-induced I_Ca suppression by preventing intracellular H⁺ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca²⁺-dependent inhibition of the I_Ca during FP exposure.     

Changes in the calcium current among different transmural regions contributes to action potential heterogeneity in rat heart

To clarify the transmural heterogeneity of action potential (AP) time course, we examined the regulation of L-type Ca²⁺ current (I_Ca,L) by voltage and Ca²⁺-dependent mechanisms. Currents were recorded using patch clamp of single rat subepicardial (EPI) and subendocardial (ENDO) of left ventricular, right ventricular (RV) and septal (SEP) cardiomyocytes. Voltage clamp commands were derived from ENDO and EPI APs or rectangular voltage pulses.During rectangular pulses, peak I_Ca,L was significantly greater in EPI than in other cells. The inactivation of I_Ca,L by Ca²⁺-dependent mechanisms (suppressed by ryanodine and BAPTA) was present in all cells but greater in extent in ENDO and SEP cells. Activation and inactivation curves for all regions show subtle differences that are Ca²⁺ sensitive, with Ca²⁺ inactivation shifting the activation variables negative by ∼ 7 mV and inactivation variables positive by 2-7 mV (EPI being least, RV greatest). In AP-clamps, the peak I_Ca,L was significantly smaller in ENDO than in EPI cells, while the integrated current was significantly larger in ENDO than in EPI cells. The results are discussed with regard to the interplay of AP time course and net Ca²⁺ influx.     

Cardiac action potential duration and calcium regulation in males and females

Adult women have longer QT intervals compared with men of a similar age, indicating differences in the speed of repolarisation of the ventricles. We investigate the influences of gender on ventricular electrophysiology and intracellular Ca²⁺ regulation of the guinea pig heart. Comparing sexually mature animals, females exhibited a significantly longer APD. Peak L-type Ca²⁺ current (I_CaL) was larger in females and when this current was inhibited with nifedipine the gender differences in APD were removed. APD differences also disappeared when the SR was depleted of Ca²⁺. Inactivation of I_CaL during a clamp step is faster in females but slower during an action potential and SR Ca²⁺ content is larger. We suggest that gender differences in APD result from variation in the kinetics of I_CaL stemming from alterations to Ca²⁺ release.     

Intracellular Cs+ activates the PKA pathway,revealing a fast,reversible, Ca2+-dependent inactivation of L-type Ca2+ current

Inactivation of the L-type Ca²⁺ current (I_CaL) was studied in isolated guinea pig ventricular myocytes with different ionic solutions. Under basal conditions, I_CaL of 82% of cells infused with Cs⁺-based intracellular solutions showed enhanced amplitude with multiphasic decay and diastolic depolarization-induced facilitation. The characteristics of I_CaL in this population of cells were not due to contamination by other currents or an artifact. These phenomena were reduced by ryanodine, caffeine, cyclopiazonic acid, the protein kinase A inhibitor H-89, and the cAMP-dependent protein kinase inhibitor. Forskolin and isoproterenol increased I_CaL by only 60% in these cells. Cells infused with either N-methyl-D-glucamine or K⁺-based intracellular solutions did not show multiphasic decay or facilitation under basal conditions. Isoproterenol increased I_CaL by 200% in these cells. In conclusion, we show that multiphasic inactivation of I_CaL is due to Ca²⁺-dependent inactivation that is reversible on a time scale of tens of milliseconds. Cs⁺ seems to activate the cAMP-dependent protein kinase pathway when used as a substitute for K⁺ in the pipette solution. L-type calcium current; calcium-dependent inactivation; facilitation; phosphorylation; cesium     

Regulation of Ca2+ signaling by acute hypoxia and acidosis in cardiomyocytes derived from human induced pluripotent stem cells

AimsThe effects of acute (100 s) hypoxia and/or acidosis on Ca²⁺ signaling parameters of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are explored here for the first time.Methods and results1) hiPSC-CMs express two cell populations: rapidly-inactivating I_Ca myocytes (τ_i<40 ms, in 4–5 day cultures) and slowly-inactivating I_Ca (τ_i ≥ 40 ms, in 6–8 day cultures). 2) Hypoxia suppressed I_Ca by 10–20% in rapidly- and 40–55% in slowly-inactivating I_Ca cells. 3) Isoproterenol enhanced I_Ca in hiPSC-CMs, but either enhanced or did not alter the hypoxic suppression. 4) Hypoxia had no differential suppressive effects in the two cell-types when Ba²⁺ was the charge carrier through the calcium channels, implicating Ca²⁺-dependent inactivation in O₂ sensing. 5) Acidosis suppressed I_Ca by ∼35% and ∼25% in rapidly and slowly inactivating I_Ca cells, respectively. 6) Hypoxia and acidosis suppressive effects on Ca-transients depended on whether global or RyR2-microdomain were measured: with acidosis suppression was ∼25% in global and ∼37% in RyR2 Ca²⁺-microdomains in either cell type, whereas with hypoxia suppression was ∼20% and ∼25% respectively in global and RyR2-microdomaine in rapidly and ∼35% and ∼45% respectively in global and RyR2-microdomaine in slowly-inactivating cells.ConclusionsVariability in I_Ca inactivation kinetics rather than cellular ancestry seems to underlie the action potential morphology differences generally attributed to mixed atrial and ventricular cell populations in hiPSC-CMs cultures. The differential hypoxic regulation of Ca²⁺-signaling in the two-cell types arises from differential Ca²⁺-dependent inactivation of the Ca²⁺-channel caused by proximity of Ca²⁺-release stores to the Ca²⁺ channels.     

Phospholemman Modulates the Gating of Cardiac L-Type Calcium Channels

Ca²⁺ entry through L-type calcium channels (Ca_V1.2) is critical in shaping the cardiac action potential and initiating cardiac contraction. Modulation of Ca_V1.2 channel gating directly affects myocyte excitability and cardiac function. We have found that phospholemman (PLM), a member of the FXYD family and regulator of cardiac ion transport, coimmunoprecipitates with Ca_V1.2 channels from guinea pig myocytes, which suggests PLM is an endogenous modulator. Cotransfection of PLM in HEK293 cells slowed Ca_V1.2 current activation at voltages near the threshold for activation, slowed deactivation after long and strong depolarizing steps, enhanced the rate and magnitude of voltage-dependent inactivation (VDI), and slowed recovery from inactivation. However, Ca²⁺-dependent inactivation was not affected. Consistent with slower channel closing, PLM significantly increased Ca²⁺ influx via Ca_V1.2 channels during the repolarization phase of a human cardiac action potential waveform. Our results support PLM as an endogenous regulator of Ca_V1.2 channel gating. The enhanced VDI induced by PLM may help protect the heart under conditions such as ischemia or tachycardia where the channels are depolarized for prolonged periods of time and could induce Ca²⁺ overload. The time and voltage-dependent slowed deactivation could represent a gating shift that helps maintain Ca²⁺ influx during the cardiac action potential waveform plateau phase.     

Protons and calcium alter gating of the hyperpolarization-activated cation current (Ih) in rod photoreceptors

We investigated the effects of protons and calcium ions on the voltage-dependent gating of the hyperpolarization-activated, nonselective cation channel current, I_h, in rod photoreceptors. I_h is a cesium-sensitive current responsible for the peak-plateau sag during the rod response to bright light. The voltage dependence of I_h activation shifted about 5 mV per pH unit, with external acidification producing positive shifts and alkalinization producing negative shifts. Increasing external [Ca²⁺] from 3 to 20 mM resulted in a large (∼17 mV) positive shift in I_h activation. External [Ca²⁺] (20 mM) blocked pH-induced shifts in activation. Cytoplasmic acidification produced by 25 mM sodium acetate led to a negative shift in inactivation (−9 mV) and internal alkalinization produced with 20 mM ammonium chloride resulted in a positive shift (+6 mV). Surface charge binding and screening theory (Gouy-Chapman-Stern) accounted for the observed shifts in I_h activation, with the best fit achieved when protons and calcium ions were assumed to bind to distinct sites on the membrane. Since light induces changes in the retinal ionic environment, these results permit us to gauge the degree to which rod light responses could be modified via alterations in I_h activation.     

Mechanism of Ca2+-dependent Inactivation of L-type Ca2+ Channels in GH3 Cells: Direct Evidence Against Dephosphorylation by Calcineurin

Dephosphorylation of Ca²⁺ channels by the Ca²⁺-activated phosphatase 2B (calcineurin) has been previously suggested as a mechanism of Ca²⁺-dependent inactivation of Ca²⁺ current in rat pituitary tumor (GH₃) cells. Although recent evidence favors an inactivation mechanism involving direct binding of Ca²⁺ to the channel protein, the alternative ``calcineurin hypothesis' has not been critically tested using the specific calcineurin inhibitors cyclosporine A (CsA) or FK506 in GH₃ cells. To determine if calcineurin plays a part in the voltage- and/or Ca²⁺-dependent components of dihydropyridine-sensitive Ca²⁺ current decay, we rapidly altered the intracellular Ca²⁺ buffering capacity of GH₃ cells by flash photolysis of DM-nitrophen, a high affinity Ca²⁺ chelator. Flash photolysis induced a highly reproducible increase in the extent of Ca²⁺ current inactivation in a two-pulse voltage protocol with Ca²⁺ as the charge carrier, but had no effect when Ba²⁺ was substituted for Ca²⁺. Despite confirmation of the abundance of calcineurin in the GH₃ cells by biochemical assays, acute application of CsA or FK506 after photolysis had no effect on Ca²⁺-dependent inactivation of Ca²⁺ current, even when excess cyclophilin or FK binding protein were included in the internal solution. Prolonged preincubation of the cells with FK506 or CsA did not inhibit Ca²⁺-dependent inactivation. Similarly, blocking calmodulin activation with calmidazolium or blocking calcineurin with fenvalerate did not influence the extent of Ca²⁺-dependent inactivation after photolysis. The results provide strong evidence against Ca²⁺-dependent dephosphorylation as the mechanism of Ca²⁺ current inactivation in GH₃ cells, but support the alternative idea that Ca²⁺-dependent inactivation reflects a direct effect of intracellular Ca²⁺ on channel gating. Received: 12 August 1996/Revised: 21 October 1996     

A computational study of the role of spike broadening in synaptic facilitation of Hermissenda

Pavlovian conditioning in Hermissenda produces a decrease in voltage-dependent (I_K,A and I_Ca) and Ca²⁺-dependent (I_K,Ca) currents, and an increase in the action potential (AP) duration in type B-photoreceptors. In addition, synaptic connections between B and A photoreceptors and B photoreceptor and type I interneurons are facilitated. The increase in AP duration, produced by decreasing one or more K⁺ currents, may account for synaptic facilitation. The present study examined this issue by using a mathematical model of the B-photoreceptor and the neurosimulator SNNAP. In the model, decreasing g_K,A by 70% increased the duration of the AP in the terminal by 41% and Ca²⁺ influx by 30%. However, if the decrease in g_K,A was combined with a decrease in g_Ca, similar to what has been reported experimentally, the Ca²⁺ influx decreased by 54%. Therefore, the concomitant change in I_Ca counter-acted the broadening-induced increase in Ca²⁺ influx in the synaptic terminal. This result suggests that a spike-duration independent process must contribute to the synaptic facilitation observed following Pavlovian conditioning.     

Functional properties of the Cav1.2 calcium channel activated by calmodulin in the absence of α2δ subunits

Voltage-activated Ca_v1.2 calcium channels require association of the pore-forming α_1C subunit with accessory Ca_vβ and α₂δ subunits. Binding of a single calmodulin (CaM) to α_1C supports Ca²⁺-dependent inactivation (CDI). The human Ca_v1.2 channel is silent in the absence of Ca_vβ and/or α₂δ. Recently, we found that coexpression of exogenous CaM (CaM_ex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of Ca_vβ. Here we discovered that CaM_ex and its Ca²⁺-insensitive mutant (CaM₁₂₃₄) rendered active α_1C/Ca_vβ channel in the absence of α₂δ. Coexpression of CaM_ex with α_1C and β_2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by ≈ +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca²⁺) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaM_ex and CaM₁₂₃₄ accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal Ca_v1.2 channel. The data suggest a previously unknown action of CaM that in the presence of Ca_vβ translates into activation of the α₂δ-deficient calcium channel and alteration of its properties.     

Ion-dependent Inactivation of Barium Current through L-type Calcium Channels

It is widely believed that Ba²⁺ currents carried through L-type Ca²⁺ channels inactivate by a voltage- dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating currents of rabbit cardiac Ca²⁺ channels expressed in different subunit combinations in tsA201 cells, we found a phase of Ba²⁺ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, I_Ba decayed as the sum of two exponentials. The slow phase (τ ≈ 6 s, 21°C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (τ ≈ 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na⁺ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba²⁺ current. The reduction of Ba²⁺ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba²⁺ entry during the pulse did not change the reversal potential for Ba²⁺. A simple model of Ca²⁺-dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1993. J. Gen. Physiol. 102:1005–1030) robustly accounts for fast Ba²⁺ current decay assuming the affinity of the inactivation site on the α₁ subunit to be 100 times lower for Ba²⁺ than Ca²⁺.     

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABAA receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA-and muscimol (Mus)-activated currents (I_gaba and I_Mus), respectively in the Mg²⁺-free external solution containing 1 μmol/L glycine at a holding potential (V _H ) of −40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 γmol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of I_gaba; (ii) when the neurons were incubated in a Ca²⁺-free bath or pre-loaded with a membrane-permeable Ca²⁺ chelator, BAPTA AM (10 μmol/L), the inhibitory effect of NMDA on I_GAba disappeared. Cd²⁺ (10 μmol/L) or La³⁺ (30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of I_gaba by NMDA application; (iii) the suppression of I_GAba by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca²⁺-dependent and CaMKII is involved in the process of the Ca²⁺-dependent inhibition.     

Multiple timescale mixed bursting dynamics in a respiratory neuron model

Experimental results in rodent medullary slices containing the pre-Bötzinger complex (pre-BötC) have identified multiple bursting mechanisms based on persistent sodium current (I _NaP) and intracellular Ca²⁺. The classic two-timescale approach to the analysis of pre-BötC bursting treats the inactivation of I _NaP, the calcium concentration, as well as the Ca²⁺-dependent inactivation of IP ₃ as slow variables and considers other evolving quantities as fast variables. Based on its time course, however, it appears that a novel mixed bursting (MB) solution, observed both in recordings and in model pre-BötC neurons, involves at least three timescales. In this work, we consider a single-compartment model of a pre-BötC inspiratory neuron that can exhibit both I _NaP and Ca²⁺ oscillations and has the ability to produce MB solutions. We use methods of dynamical systems theory, such as phase plane analysis, fast-slow decomposition, and bifurcation analysis, to better understand the mechanisms underlying the MB solution pattern. Rather surprisingly, we discover that a third timescale is not actually required to generate mixed bursting solutions. Through our analysis of timescales, we also elucidate how the pre-BötC neuron model can be tuned to improve the robustness of the MB solution.     

Mechanistic Investigation of the Arrhythmogenic Role of Oxidized CaMKII in the Heart

Oxidative stress and calcium (Ca²⁺)/calmodulin (CaM)-dependent protein kinase II (CaMKII) both play important roles in the pathogenesis of cardiac disease. Although the pathophysiological relevance of reactive oxygen species (ROS) and CaMKII has been appreciated for some time, recent work has shown that ROS can directly oxidize CaMKII, leading to its persistent activity and an increase of the likelihood of cellular arrhythmias such as early afterdepolarizations (EADs). Because CaMKII modulates the function of many proteins involved in excitation-contraction coupling, elucidation of its role in cardiac function, in both healthy and oxidative stress conditions, is challenging. To investigate this role, we have developed a model of CaMKII activation that includes both the phosphorylation-dependent and the newly identified oxidation-dependent activation pathways. This model is incorporated into our previous local-control model of the cardiac myocyte that describes excitation-contraction coupling via stochastic simulation of individual Ca²⁺ release units and CaMKII-mediated phosphorylation of L-type Ca²⁺ channels (LCCs), ryanodine receptors and sodium (Na⁺) channels. The model predicts the experimentally measured slow-rate dependence of H₂O₂-induced EADs. Upon increased H₂O₂, simulations suggest that selective activation of late Na⁺ current (I_NaL), although it prolongs action potential duration, is not by itself sufficient to produce EADs. Similar results are obtained if CaMKII effects on LCCs and ryanodine receptors are considered separately. However, EADs emerge upon simultaneous activation of both LCCs and Na⁺ channels. Further modeling results implicate activation of the Na⁺-Ca²⁺ exchanger (NCX) as an important player in the generation of EADs. During bradycardia, the emergence of H₂O₂-induced EADs was correlated with a shift in the timing of NCX current reversal toward the plateau phase earlier in the action potential. Using the timing of NCX current reversal as an indicator event for EADs, the model identified counterintuitive ionic changes—difficult to experimentally dissect—that have the greatest influence on ROS-related arrhythmia propensity.     

Effects of Copper on T-Type Ca<Superscript>2+</Superscript> Channels in Mouse Spermatogenic Cells

Low voltage-activated, rapidly inactivating T-type Ca²⁺ channels are found in a variety of cells, where they regulate electrical activity and Ca²⁺ entry. In whole-cell patch-clamp recordings from mouse spermatogenic cells, trace element copper (Cu²⁺) inhibited T-type Ca²⁺ current (I _T-Ca) with IC₅₀ of 12.06 μM. Inhibition of I _T-Ca by Cu²⁺ was concentration-dependent and mildly voltage-dependent. When voltage stepped to −20 mV, Cu²⁺ (10 μM) inhibited I _T-Ca by 49.6 ± 4.1%. Inhibition of I _T-Ca by Cu²⁺ was accompanied by a shift of −2.23 mV in the voltage dependence of steady-state inactivation. Cu²⁺ upshifted the current–voltage (I-V) curve. To know the change of the gating kinetics of T-type Ca²⁺ channels, we analyzed the effect of Cu²⁺ on activation, inactivation, deactivation and reactivation of T-type Ca²⁺ channels. Since T-type Ca²⁺ channels are a key component in capacitation and the acrosome reaction, our data suggest that Cu²⁺ can affect male reproductive function through T-type Ca²⁺ channels as a preconception contraceptive material.