Rate Of Action Potential Repolarization Research Articles

Channel sialic acids limit hERG channel activity during the ventricular action potential

Activity of human ether-a-go-go-related gene (hERG) 1 voltage-gated K(+) channels is responsible for portions of phase 2 and phase 3 repolarization of the human ventricular action potential. Here, we questioned whether and how physiologically and pathophysiologically relevant changes in surface N-glycosylation modified hERG channel function. Voltage-dependent hERG channel gating and activity were evaluated as expressed in a set of Chinese hamster ovary (CHO) cell lines under conditions of full glycosylation, no sialylation, no complex N-glycans, and following enzymatic deglycosylation of surface N-glycans. For each condition of reduced glycosylation, hERG channel steady-state activation and inactivation relationships were shifted linearly by significant depolarizing ∼9 and ∼18 mV, respectively. The hERG window current increased significantly by 50-150%, and the peak shifted by a depolarizing ∼10 mV. There was no significant change in maximum hERG current density. Deglycosylated channels were significantly more active (20-80%) than glycosylated controls during phases 2 and 3 of action potential clamp protocols. Simulations of hERG current and ventricular action potentials corroborated experimental data and predicted reduced sialylation leads to a 50-70-ms decrease in action potential duration. The data describe a novel mechanism by which hERG channel gating is modulated through physiologically and pathophysiologically relevant changes in N-glycosylation; reduced channel sialylation increases hERG channel activity during the action potential, thereby increasing the rate of action potential repolarization.

N-Glycans Modulate hERG1A Window Current

Voltage-gated K+ channels are primarily responsible for the repolarization phases of neuronal, skeletal and cardiac muscle action potentials. Activity of the human ether-a-go-go-related gene 1 (hERG1) voltage-gated K+ channel produces a K+ current responsible for much of late phase II and phase III repolarization of the human cardiac action potential. HERG1A has two putative N-glycosylation sites located in the S5-S6 linker region, one of which is N-glycosylated. The aim of this study was to determine whether and how N-linked glycosylation modifies hERG1A channel function. Voltage-dependent gating of hERG1A were evaluated under conditions of full glycosylation, no sialylation, in the absence of complex N-glycans, and following the removal of the full N-glycosylation structure. The hERG1A steady-state activation relationships were shifted along the voltage axis by a significant, depolarizing ∼9 mV under each condition of reduced glycosylation. Steady state channel availability curves were shifted by a much greater depolarizing 20-30 mV under conditions of reduced glycosylation. The depolarizing, non-uniform shifts in voltage-dependent steady state activation and inactivation caused a large rightward shift and an increase in the hERG1A window current. This suggests that reduced glycosylation caused an increase in the persistent hERG current that occurs at more depolarized potentials as observed. This increase and shift in window current would lead to increased hERG1A activity during the action potential, effectively increasing the rate of repolarization, and reducing AP duration, as predicted through in silico modeling of the ventricular AP. Overall, these data indicate a functional role for N-glycosylation in the modulation of hERG1A channel activity, suggesting that even small changes in channel N-glycosylation modulate hERG1A activity, and thereby likely impact the rate of action potential repolarization.

Metabotropic glutamate receptors 1 and 5 differentially regulate bulbar dopaminergic cell function

Effects of activation of metabotropic glutamatergic receptors (mGluR) were investigated in mouse dopaminergic olfactory bulb neurons. After blockage of ionotropic receptors, focal application of glutamate or of group I/II mGluR agonist t-ACPD resulted in a depolarization, paralleled by an inward current in voltage-clamp conditions. The Group I agonist DHPG induced a depolarization, which could be largely blocked by mGluR1 antagonists. The DHPG action i) was prevented by buffering intracellular Ca 2+ with BAPTA and by a phospholipase C inhibitor; ii) was not affected by the block of Ca 2+ entry, and iii) was blocked by inhibitors of the Na +/Ca 2+ exchanger. These observations were interpreted as a mGluR1-mediated intracellular Ca 2+ release, followed by the activation of an electrogenic Na +/Ca 2+ exchanger. The mGluR5 agonist CHPG induced a hyperpolarization of membrane potential, resulting in a decrease of the spontaneous firing frequency. CHPG induced i) a decrease in membrane resistance; ii) an increase in the action potential repolarization rate, and iii) an increase in the amplitude of the afterhyperpolarization. This was interpreted as a mGluR5-mediated opening of a K + conductance. These data suggest that mGluR1 and mGluR5 play different and non-overlapping roles in the regulation of the excitability of bulbar dopaminergic neurons.

Mechanism of Increased BK Channel Activation from a Channel Mutation that Causes Epilepsy

Concerted depolarization and Ca2+ rise during neuronal action potentials activate large-conductance Ca2+- and voltage-dependent K+ (BK) channels, whose robust K+ currents increase the rate of action potential repolarization. Gain-of-function BK channels in mouse knockout of the inhibitory β4 subunit and in a human mutation (αD434G) have been linked to epilepsy. Here, we investigate mechanisms underlying the gain-of-function effects of the equivalent mouse mutation (αD369G), its modulation by the β4 subunit, and potential consequences of the mutation on BK currents during action potentials. Kinetic analysis in the context of the Horrigan-Aldrich allosteric gating model revealed that changes in intrinsic and Ca2+-dependent gating largely account for the gain-of-function effects. D369G causes a greater than twofold increase in the closed-to-open equilibrium constant (6.6e−7→1.65e−6) and an approximate twofold decrease in Ca2+-dissociation constants (closed channel: 11.3→5.2 µM; open channel: 0.92→0.54 µM). The β4 subunit inhibits mutant channels through a slowing of activation kinetics. In physiological recording solutions, we established the Ca2+ dependence of current recruitment during action potential–shaped stimuli. D369G and β4 have opposing effects on BK current recruitment, where D369G reduces and β4 increases K1/2 (K1/2 μM: αWT 13.7, αD369G 6.3, αWT/β4 24.8, and αD369G/β4 15.0). Collectively, our results suggest that the D369G enhancement of intrinsic gating and Ca2+ binding underlies greater contributions of BK current in the sharpening of action potentials for both α and α/β4 channels.

Mechanism Of Increased Bk Channel Activation From A Channel Mutation That Causes Epilepsy

Concerted depolarization and calcium rise during action potentials activate large-conductance calcium- and voltage- activated (BK) potassium channels, whose robust potassium currents increase the rate of action potential repolarization. Gain-of-function BK channels, both in mouse knockout of the inhibitory β4 subunit, and in humans with (αD434G) mutation have been linked to epilepsy. Here, we investigate mechanisms underlying the gain of function effects of the equivalent mouse mutation (αD370G), its modulation by the β4 subunit and potential consequences of the mutation on BK currents during action potentials. Kinetic analysis in the context of the Horrigan-Aldrich allosteric gating model revealed that changes in intrinsic and calcium-dependent gating largely account for the gain-of-function effects. D370G causes a greater than 2-fold increase in intrinsic gating equilibrium constant (1.65e-6 versus 6.6e-7) and an approximately 2-fold decrease in calcium dissociation constants (closed channel: 5.2 versus 11.3 μM, open channel: 0.54 versus 0.92 μM). Contrary to a previous report, co-expression of β4 produced similar changes in G-V relationships and gating kinetics for wildtype and mutant channels, suggesting that αD370G channels can be inhibited by β4. In physiological recording solutions, we established calcium dependence of BK current recruitment during action potential-shaped stimuli. D370G reduces K1/2 for both α (6.3 versus 13.7 μM) and α/β4 (15.0 versus 24.8 μM) channels. Although increased recruitment of BK currents by the mutation for both channel types are highly calcium dependent, greater effects were observed for the α/β4 BK channels. These results suggest that the D370G enhancement of intrinsic gating and apparent calcium affinity allow a greater contribution of BK current in sharpening of action potentials both in the presence and absence of the inhibitory β4 subunit.

Differential expression of calcium channels in sympathetic and parasympathetic preganglionic inputs to neurons in paracervical ganglia of guinea-pigs

Neurons in pelvic ganglia receive nicotinic excitatory post-synaptic potentials (EPSPs) from sacral preganglionic neurons via the pelvic nerve, lumbar preganglionic neurons via the hypogastric nerve or both. We tested the effect of a range of calcium channel antagonists on EPSPs evoked in paracervical ganglia of female guinea-pigs after pelvic or hypogastric nerve stimulation. ω-Conotoxin GVIA (CTX GVIA, 100 nM) or the novel N-type calcium channel antagonist, CTX CVID (100 nM) reduced the amplitude of EPSPs evoked after pelvic nerve stimulation by 50–75% but had no effect on EPSPs evoked by hypogastric nerve stimulation. Combined addition of CTX GVIA and CTX CVID was no more effective than either antagonist alone. EPSPs evoked by stimulating either nerve trunk were not inhibited by the P/Q calcium channel antagonist, ω-agatoxin IVA (100 nM), nor the L-type calcium channel antagonist, nifedipine (30 μM). SNX 482 (300 nM), an antagonist at some R-type calcium channels, inhibited EPSPs after hypogastric nerve stimulation by 20% but had little effect on EPSPs after pelvic nerve stimulation. Amiloride (100 μM) inhibited EPSPs after stimulation of either trunk by 40%, while nickel (100 μM) was ineffective. CTX GVIA or CTX CVID (100 nM) also slowed the rate of action potential repolarization and reduced afterhyperpolarization amplitude in paracervical neurons. Thus, release of transmitter from the terminals of sacral preganglionic neurons is largely dependent on calcium influx through N-type calcium channels, although an unknown calcium channel which is resistant to selective antagonists also contributes to release. Release of transmitter from lumbar preganglionic neurons does not require calcium entry through either conventional N-type calcium channels or the variant CTX CVID-sensitive N-type calcium channel and seems to be mediated largely by a novel calcium channel.

Wheel-running exercise alters rat diaphragm action potentials and their regulation by K+ channels.

Endurance exercise modifies regulatory systems that control skeletal muscle Na+ and K+ fluxes, in particular Na+-K+-ATPase-mediated transport of these ions. Na+ and K+ ion channels also play important roles in the regulation of ionic movements, specifically mediating Na+ influx and K+ efflux that occur during contractions resulting from action potential depolarization and repolarization. Whether exercise alters skeletal muscle electrophysiological properties controlled by these ion channels is unclear. The present study tested the hypothesis that endurance exercise modifies diaphragm action potential properties. Exercised rats spent 8 wk with free access to running wheels, and they were compared with sedentary rats living in conventional rodent housing. Diaphragm muscle was subsequently removed under anesthesia and studied in vitro. Resting membrane potential was not affected by endurance exercise. Muscle from exercised rats had a slower rate of action potential repolarization than that of sedentary animals (P = 0.0098), whereas rate of depolarization was similar in the two groups. The K+ channel blocker 3,4-diaminopyridine slowed action potential repolarization and increased action potential area of both exercised and sedentary muscle. However, these effects were significantly smaller in diaphragm from exercised than sedentary rats. These data indicate that voluntary running slows diaphragm action potential repolarization, most likely by modulating K+ channel number or function.

Slowing of rat diaphragm action potential depolarization by endurance treadmill training

This study tested the hypothesis that the action potential properties of the diaphragm muscle are altered by endurance exercise treadmill training. Rats underwent treadmill running or sham training for 8 weeks, and intracellular electrophysiological recordings were subsequently performed in vitro. Diaphragm resting membrane potential was not altered by training. The maximal rate of action potential depolarization was reduced significantly by exercise training, from 551±16 to 445±15 mV/ms ( P<0.00002). In contrast the rate of action potential repolarization was not significantly different between the two groups ( P=0.25). Action potential height was significantly higher in control compared with trained muscle (84.5±1.0 vs. 78.4±1.2 mV, P<0.0005). The combination of slowed action depolarization and decreased peak action potential height resulted in no net change in action potential area. Thus treadmill running endurance exercise training slows rat diaphragm action potential depolarization but not repolarization, suggestive of altered Na + but not K + channel function.

Postnatal changes in membrane properties of mice trigeminal ganglion neurons.

Intracellular recordings from neurons in the mouse trigeminal ganglion (TG) in vitro were used to characterize changes in membrane properties that take place from early postnatal stages (P0-P7) to adulthood (>P21). All neonatal TG neurons had uniformly slow conduction velocities, whereas adult neurons could be separated according to their conduction velocity into Adelta and C neurons. Based on the presence or absence of a marked inflection or hump in the repolarization phase of the action potential (AP), neonatal neurons were divided into S- (slow) and F-type (fast) neurons. Their passive and subthreshold properties (resting membrane potential, input resistance, membrane capacitance, and inward rectification) were nearly identical, but they showed marked differences in AP amplitude, AP overshoot, AP duration, rate of AP depolarization, rate of AP repolarization, and afterhyperpolarization (AHP) duration. Adult TG neurons also segregated into S- and F-type groups. Differences in their mean AP amplitude, AP overshoot, AP duration, rate of AP depolarization, rate of AP repolarization, and AHP duration were also prominent. In addition, axons of 90% of F-type neurons and 60% of S-type neurons became faster conducting in their central and peripheral branch, suggestive of axonal myelination. The proportion of S- and F-type neurons did not vary during postnatal development, suggesting that these phenotypes were established early in development. Membrane properties of both types of TG neurons evolved differently during postnatal development. The nature of many of these changes was linked to the process of myelination. Thus myelination was accompanied by a decrease in AP duration, input resistance (R(in)), and increase in membrane capacitance (C). These properties remained constant in unmyelinated neurons (both F- and S-type). In adult TG, all F-type neurons with inward rectification were also fast-conducting Adelta, suggesting that those F-type neurons showing inward rectification at birth will evolve to F-type Adelta neurons with age. The percentage of F-type neurons showing inward rectification also increased with age. Both F- and S-type neurons displayed changes in the sensitivity of the AP to reductions in extracellular Ca(2+) or substitution with Co(2+) during the process of maturation.

Contribution of presynaptic calcium-activated potassium currents to transmitter release regulation in cultured Xenopus nerve–muscle synapses

Using Xenopus nerve–muscle co-cultures, we have examined the contribution of calcium-activated potassium (K Ca) channels to the regulation of transmitter release evoked by single action potentials. The presynaptic varicosities that form on muscle cells in these cultures were studied directly using patch-clamp recording techniques. In these developing synapses, blockade of K Ca channels with iberiotoxin or charybdotoxin decreased transmitter release by an average of 35%. This effect would be expected to be caused by changes in the late phases of action potential repolarization. We hypothesize that these changes are due to a reduction in the driving force for calcium that is normally enhanced by the local hyperpolarization at the active zone caused by potassium current through the K Ca channels that co-localize with calcium channels. In support of this hypothesis, we have shown that when action potential waveforms were used as voltage-clamp commands to elicit calcium current in varicosities, peak calcium current was reduced only when these waveforms were broadened beginning when action potential repolarization was 20% complete. In contrast to peak calcium current, total calcium influx was consistently increased following action potential broadening. A model, based on previously reported properties of ion channels, faithfully reproduced predicted effects on action potential repolarization and calcium currents. From these data, we suggest that the large-conductance K Ca channels expressed at presynaptic varicosities regulate transmitter release magnitude during single action potentials by altering the rate of action potential repolarization, and thus the magnitude of peak calcium current.

Contribution of the Selectivity Filter to Inactivation in Potassium Channels

Voltage-gated K + channels exhibit a slow inactivation process, which becomes an important influence on the rate of action potential repolarization during prolonged or repetitive depolarization. During slow inactivation, the outer mouth of the permeation pathway undergoes a conformational change. We report here that during the slow inactivation process, the channel progresses through at least three permeation states; from the initial open state that is highly selective for K +, the channel enters a state that is less permeable to K + and more permeable to Na +, and then proceeds to a state that is non-conducting. Similar results were obtained in three different voltage-gated K + channels: Kv2.1, a channel derived from Shaker (Shaker Δ A463C), and a chimeric channel derived from Kv2.1 and Kv1.3 that displays classical C-type inactivation. The change in selectivity displayed both voltage- and time-dependent properties of slow inactivation and was observed with K + on either side of the channel. Elevation of internal [K +] inhibited Na + conduction through the inactivating channel in a concentration-dependent manner. These results indicate that the change in selectivity filter function is an integral part of the slow inactivation mechanism, and argue against the hypothesis that the inactivation gate is independent from the selectivity filter. Thus, these data suggest that the selectivity filter is itself the inactivation gate.

Mechanism of action potential prolongation by RP 58866 and its active enantiomer, terikalant. Block of the rapidly activating delayed rectifier K+ current, IKr.

The class III antiarrhythmic agent RP 58866 and its active enantiomer, terikalant, are reported to selectively block the inward rectifier K+ current, IK1. These drugs have demonstrated efficacy in animal models of cardiac arrhythmias, suggesting that block of IK1 may be a useful antiarrhythmic mechanism. The symmetrical action potential (AP)-prolonging and bradycardic effects of these drugs, however, are inconsistent with a sole effect on IK1. We studied the effects of RP 58866 and terikalant on AP and outward K+ currents in guinea pig ventricular myocytes. RP 58866 and terikalant potently blocked the rapidly activating delayed rectifier K+ current, IKr, with IC50S of 22 and 31 nmol/L, respectively. Block of IK1 was approximately 250-fold less potent; IC50S were 8 and 6 mumol/L, respectively. No significant block of the slowly activating delayed rectifier, IK1, was observed at < or = 10 mumol/L. The phenotypical IKr currents in mouse AT-1 cells and Xenopus oocytes expressing HERG were also blocked 50% by 200 to 250 nmol/L RP 58866 or terikalant, providing further conclusive evidence for potent block of IKr. RP 58866 < or = 1 mumol/L and dofetilide increased AP duration symmetrically, consistent with selective block of IKr. Only higher concentrations (> or = 10 mumol/L) of RP 58866 slowed the rate of AP repolarization and decreased resting membrane potential, consistent with an additional but substantially less potent block of IK1. These data demonstrate that RP 58866 and terikalant are potent blockers of IKr and prompt a reinterpretation of previous studies that assumed specific block of IK1 by these drugs.

318 Induction of inward current by the activation of mglur1 type receptor of mouse cerebellar purkinje cells

Effects of activation of metabotropic glutamatergic receptors (mGluR) were investigated in mouse dopaminergic olfactory bulb neurons. After blockage of ionotropic receptors, focal application of glutamate or of group I/II mGluR agonist t-ACPD resulted in a depolarization, paralleled by an inward current in voltage-clamp conditions. The Group I agonist DHPG induced a depolarization, which could be largely blocked by mGluR1 antagonists. The DHPG action i) was prevented by buffering intracellular Ca2+ with BAPTA and by a phospholipase C inhibitor; ii) was not affected by the block of Ca2+ entry, and iii) was blocked by inhibitors of the Na+/Ca2+ exchanger. These observations were interpreted as a mGluR1-mediated intracellular Ca2+ release, followed by the activation of an electrogenic Na+/Ca2+ exchanger. The mGluR5 agonist CHPG induced a hyperpolarization of membrane potential, resulting in a decrease of the spontaneous firing frequency. CHPG induced i) a decrease in membrane resistance; ii) an increase in the action potential repolarization rate, and iii) an increase in the amplitude of the afterhyperpolarization. This was interpreted as a mGluR5-mediated opening of a K+ conductance. These data suggest that mGluR1 and mGluR5 play different and non-overlapping roles in the regulation of the excitability of bulbar dopaminergic neurons.

Potassium currents contributing to action potential repolarization and the afterhyperpolarization in rat vagal motoneurons.

1. Intracellular recordings were made from neurons in the dorsal motor nucleus of the vagus (DMV) in transverse slices of rat medulla maintained in vitro at 30 degrees C. Neurons had a resting potential of -59.8 +/- 1.4 (SE) mV (n = 39) and input resistance of 293 +/- 23 M omega (n = 44). 2. Depolarization elicited overshooting action potentials that were blocked by tetrodotoxin (TTX; 1 microM). In the presence of TTX, two types of action potentials having low and high thresholds could be elicited. The action potentials were blocked by cobalt (2 mM) indicating they were mediated by calcium currents. 3. Under voltage clamp, depolarization of the cell from membrane potentials negative of the resting potential activated a transient potassium current. This current was selectively blocked by 4-aminopyridine (4-AP) (5 mM) and catechol (5 mM) indicating that it is an A-type current. This current inactivated with a time constant of 420 ms and recovered from inactivation with a time constant of 26 ms. 4. When calcium currents were blocked by cadmium or cobalt, the rate of action potential repolarization was slower. In the presence of tetraethylammonium (TEA; 200-400 microM) or charybdotoxin (CTX; 30 nM) a small "hump" appeared on the repolarizing phase of the action potential that was abolished by addition of cadmium. These results indicate that a calcium-activated potassium current (IC) contributes to action potential repolarization. 5. Actions potentials elicited from hyperpolarized membrane potentials repolarized faster than those elicited from resting membrane potential. This effect could be blocked by catechol, indicating that voltage-dependent potassium currents (IA) can also contribute to action-potential repolarization. In the presence of catechol and calcium channel blockers, action potentials still had a significant early afterhyperpolarization suggesting that another calcium independent outward current is also active during repolarization. This fast afterhyperpolarizations (AHP) was not blocked by TEA. 6. Action potentials were followed by prolonged AHPs, which had two phases. The initial part of the AHP was blocked by apamin (100 nM) indicating that it results from activation of SK type calcium-activated potassium channels. The slow phase was selectively blocked by catechol suggesting that it is due to activation of IA. 7. It is concluded that a TTX-sensitive sodium current and two calcium currents contribute to the action potential in rat DMV neurons. At least three different currents contribute to action-potential repolarization: IC, IA, and a third unidentified calcium-insensitive outward current.(ABSTRACT TRUNCATED AT 400 WORDS)

Two transient potassium currents in layer V pyramidal neurones from cat sensorimotor cortex.

1. Two transient outward currents were identified in large pyramidal neurones from layer V of cat sensorimotor cortex ('Betz cells') using an in vitro brain slice preparation and single-microelectrode voltage clamp. Properties of the currents deduced from voltage-clamp measurements were reflected in neuronal responses during constant current stimulation. 2. Both transient outward currents rose rapidly after a step depolarization, but their subsequent time course differed greatly. The fast-transient current decayed within 20 ms, while the slow-transient current took greater than 10 s to decay. Raised extracellular potassium reduced current amplitude. Both currents were present in cadmium-containing or calcium-free perfusate. 3. Tetraethylammonium had little effect on the slow-transient current at a concentration of 1 mM, but the fast-transient current was reduced by 60%. 4-Aminopyridine had little effect on the fast-transient current over the range 20 microM-2 mM, but these concentrations reduced the slow-transient current and altered its time course. 4. Both transient currents were evoked by depolarizations below action potential threshold. The fast-transient current was evoked by a 7 mV smaller depolarization than the slow-transient current, but its chord conductance increased less steeply with depolarization. 5. Voltage-dependent inactivation of the fast-transient was steeper than that of the slow-transient current (4 vs. 7 mV per e-fold change), and half-inactivation occurred at a less negative potential (-59 vs. -65 mV). The activation and inactivation characteristics of each current overlapped, however, implying the existence of a steady 'window current' extending over a range of approximately 14 mV beginning negative to action potential threshold. 6. The fast-transient current displayed a clear voltage dependence of both its activation and inactivation kinetics, whereas the slow-transient current did not. Recovery of either current from inactivation took about 1 s near -70 mV. The recovery of the slow-transient current became faster with hyperpolarization. 7. The contribution of each transient current to repolarization of the action potential was assessed from pharmacological responses. Blockade of calcium influx had little or no effect on the rate of action potential repolarization, whereas the selective reduction of either transient current caused significant slowing of repolarization. 8. We conclude that Betz cells possess at least two transient potassium currents, each a member of the rapidly expanding family of voltage-gated potassium currents that have been identified in various cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Action potential repolarization may involve a transient, Ca2+-sensitive outward current in a vertebrate neurone.

Repolarization of the action potential in squid axon1 and several types of neurones2-4 involves a voltage-activated potassium (K+) current. Voltage clamp analysis has demonstrated that this current has rapid activation kinetics1,3-5. In several neuronal types, the same technique has also revealed a slowly activated K+ current that is calcium (Ca2+)-sensitive3,5-10. This slow Ca2+-activated K+ current is the major current underlying the late, slower portion of the after-hyperpolarization following an action potential11-14. In several muscle types, fast, transient Ca2+-dependent K+ currents have been described15-17 which may contribute to repolarization of the action potential. Rapidly activating, Ca2+-dependent K+ currents have been observed in sympathetic neurones of the bullfrog and it has been suggested that they contribute to action potential repolarization of those neurones8,9,18. We have studied the membrane currents in bullfrog sympathetic neurones using voltage clamp methods and report here a transient outward current that appears to be composed of two separate currents. One of those currents is a transient, Ca2+-sensitive outward current as indicated by a significant reduction of the current by treatments that reduce or block Ca2+ entry (Mn2+, Cd2+, Co2+, Mg2+ or Ca2+-free Ringer). Such treatments also decreased the rate of action potential repolarization. The results suggest that this current is involved in repolarization of the action potential and consequently may regulate Ca2+ entry into the neurone during spike activity.