Voltage Dependence Of Channel Inactivation Research Articles

Structural modeling of the hERG potassium channel and associated drug interactions.

The voltage-gated potassium channel, KV11.1, encoded by the human Ether-à-go-go-Related Gene (hERG), is expressed in cardiac myocytes, where it is crucial for the membrane repolarization of the action potential. Gating of the hERG channel is characterized by rapid, voltage-dependent, C-type inactivation, which blocks ion conduction and is suggested to involve constriction of the selectivity filter. Mutations S620T and S641A/T within the selectivity filter region of hERG have been shown to alter the voltage dependence of channel inactivation. Because hERG channel blockade is implicated in drug-induced arrhythmias associated with both the open and inactivated states, we used Rosetta to simulate the effects of hERG S620T and S641A/T mutations to elucidate conformational changes associated with hERG channel inactivation and differences in drug binding between the two states. Rosetta modeling of the S641A fast-inactivating mutation revealed a lateral shift of the F627 side chain in the selectivity filter into the central channel axis along the ion conduction pathway and the formation of four lateral fenestrations in the pore. Rosetta modeling of the non-inactivating mutations S620T and S641T suggested a potential molecular mechanism preventing F627 side chain from shifting into the ion conduction pathway during the proposed inactivation process. Furthermore, we used Rosetta docking to explore the binding mechanism of highly selective and potent hERG blockers - dofetilide, terfenadine, and E4031. Our structural modeling correlates well with much, but not all, existing experimental evidence involving interactions of hERG blockers with key residues in hERG pore and reveals potential molecular mechanisms of ligand interactions with hERG in an inactivated state.

The potassium channel auxiliary subunit Kvβ2 (Kcnab2) regulates Kv1 channels and dopamine neuron firing.

Ion channel complexes typically consist of both pore-forming subunits and auxiliary subunits that do not directly conduct current but can regulate trafficking or alter channel properties. Isolating the role of these auxiliary subunits in neurons has proved difficult due to a lack of specific pharmacological agents and the potential for developmental compensation in constitutive knockout models. Here, we use cell-type-specific viral-mediated CRISPR/Cas9 mutagenesis to target the potassium channel auxiliary subunit Kvβ2 (Kcnab2) in dopamine neurons in the adult mouse brain. We find that mutagenesis of Kcnab2 reduces surface expression of Kv1.2, the primary Kv1 pore-forming subunit expressed in dopamine neurons, and shifts the voltage dependence of inactivation of potassium channel currents toward more hyperpolarized potentials. Loss of Kcnab2 broadens the action potential waveform in spontaneously firing dopamine neurons recorded in slice, reduces the afterhyperpolarization amplitude, and increases spike timing irregularity and excitability, all of which is consistent with a reduction in potassium channel current. Similar effects were observed with mutagenesis of the pore-forming subunit Kv1.2 (Kcna2). These results identify Kv1 currents as important contributors to dopamine neuron firing and demonstrate a role for Kvβ2 subunits in regulating the trafficking and gating properties of these ion channels. Furthermore, they demonstrate the utility of CRISPR-mediated mutagenesis in the study of previously difficult to isolate ion channel subunits.NEW & NOTEWORTHY Here, we utilize CRISPR/Cas9-mediated mutagenesis in dopamine neurons in mice to target the gene encoding Kvβ2, an auxiliary subunit that forms a part of Kv1 channel complexes. We find that the absence of Kvβ2 alters action potential properties by reducing surface expression of pore-forming subunits and shifting the voltage dependence of channel inactivation. This work establishes a new function for Kvβ2 subunits and Kv1 complexes in regulating dopamine neuron activity.

Pharmacological Inhibition of the Voltage-Gated Sodium Channel NaV1.7 Alleviates Chronic Visceral Pain in a Rodent Model of Irritable Bowel Syndrome.

The human nociceptor-specific voltage-gated sodium channel 1.7 (hNaV1.7) is critical for sensing various types of somatic pain, but it appears not to play a primary role in acute visceral pain. However, its role in chronic visceral pain remains to be determined. We used assay-guided fractionation to isolate a novel hNaV1.7 inhibitor, Tsp1a, from tarantula venom. Tsp1a is 28-residue peptide that potently inhibits hNaV1.7 (IC50 = 10 nM), with greater than 100-fold selectivity over hNaV1.3-hNaV1.6, 45-fold selectivity over hNaV1.1, and 24-fold selectivity over hNaV1.2. Tsp1a is a gating modifier that inhibits NaV1.7 by inducing a hyperpolarizing shift in the voltage-dependence of channel inactivation and slowing recovery from fast inactivation. NMR studies revealed that Tsp1a adopts a classical knottin fold, and like many knottin peptides, it is exceptionally stable in human serum. Remarkably, intracolonic administration of Tsp1a completely reversed chronic visceral hypersensitivity in a mouse model of irritable bowel syndrome. The ability of Tsp1a to reduce visceral hypersensitivity in a model of irritable bowel syndrome suggests that pharmacological inhibition of hNaV1.7 at peripheral sensory nerve endings might be a viable approach for eliciting analgesia in patients suffering from chronic visceral pain.

LRRC8 N termini influence pore properties and gating of volume-regulated anion channels (VRACs)

Volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have various roles in physiology and pathology. VRACs were recently discovered to be formed by heteromers of leucine-rich repeat–containing 8 (LRRC8) proteins. However, the structural determinants of VRAC permeation and gating remain largely unknown. We show here that the short stretch preceding the first LRRC8 transmembrane domain determines VRAC conductance, ion permeability, and inactivation gating. Substituted-cysteine accessibility studies revealed that several of the first 15 LRRC8 residues are functionally important and exposed to a hydrophilic environment. Substituting glutamate 6 with cysteine decreased the amplitudes of swelling-activated ICl,vol currents, strongly increased iodide-over-chloride permeability, and markedly shifted the voltage dependence of channel inactivation. Importantly, these effects were reversed by 2-sulfonatoethyl methanethiosulfonate, which restores the negative charge at this amino acid position. Cd2+-mediated blocking of ICl,vol in cysteine variants suggested that the LRRC8 N termini come close together in the multimeric channel complex and might form part of the pore. We propose a model in which the N termini of the LRRC8 subunits line the cytoplasmic portion of the VRAC pore, possibly by folding back into the ion permeation pathway.

Sodium channel Nav1.8: Emerging links to human disease.

The NaV1.8 sodium channel, encoded by gene SCN10A, was initially termed sensory neuron-specific (SNS) due to prominent expression in primary sensory neurons including dorsal root ganglion (DRG) neurons. Early studies on rodent NaV1.8 demonstrated depolarized voltage dependence of channel inactivation, a slow rate of inactivation, and rapid recovery from inactivation. As a result of these biophysical properties, NaV1.8 supports repetitive firing in response to sustained depolarization. This article reviews recent studies that reveal multiple links of NaV1.8 to human disease: (1) It has recently been shown that functional attributes that distinguish NaV1.8 from other sodium channel subtypes are exaggerated in human NaV1.8; its influence on neuronal activity is thus greater than previously thought. (2) Gain-of-function mutations of NaV1.8 that produce DRG neuron hyperexcitability have been found in 3% of patients with painful neuropathy, establishing a role in pathogenesis. (3) NaV1.8 is ectopically expressed within Purkinje neurons in multiple sclerosis (MS), where it perturbs electrical activity. Recent evidence indicates that variants of SCN10A predict the degree of cerebellar dysfunction in MS. (4) Emerging evidence has linked SCN10A variants to disorders of cardiac rhythm, via mechanisms that may include an effect on cardiac innervation. Involvement of NaV1.8 in neurologic disease may have therapeutic implications. NaV1.8-specific blocking agents, under development, ameliorate pain and attenuate MS-like deficits in animal models. Recent studies suggest that pharmacogenomics may permit the matching of specific channel blocking agents to particular patients. The new links of NaV1.8 in human disease raise new questions, but also suggest new therapeutic strategies.

A PQ-Channel Mutation Associated with Epilepsy Alters the Voltage Dependence of Channel Inactivation

Epilepsy affects 1 in 140 people, or nearly 50 million people worldwide. It is characterized by seizures that often result from neuronal hyper-excitability. A recent genome-wide study uncovered many mutations associated with epilepsy in voltage-gated calcium channels (VGCC). However, the molecular and cellular consequences of these mutations, and hence, their epileptogenic mechanisms remain unknown. Here, we investigated two mutations that occur in PQ-type voltage-gated calcium channels, which are responsible for neurotransmitter release: R477H (in the I-II loop) and Q1957X (a truncation mutation). The mutations were introduced into a human PQ channel in the pGEMHE vector. The DNA for WT or mutant channels, together with the DNA for other VGCC subunits required for proper channel expression, was subjected to in vitro cRNA synthesis. The resulting cRNA was subsequently injected into Xenopus oocytes, and channel currents were examined using Two-Electrode Voltage Clamp four days later. We thus compared the biophysical properties of WT and mutant channels. We found that the R477H mutation shifts the voltage dependence of inactivation to more depolarized voltages, rendering the mutant channels more difficult to inactivate compared to the WT channel. In addition, the time constant of inactivation was slower for the mutant. At the same time, the voltage dependence of activation was unchanged. These results suggest a potential mechanism for epileptogenesis: reduced channel inactivation would contribute to increased cellular excitability and excitotoxicity - two hallmarks of epilepsy. The results also highlight the role of the I-II loop of VGCC in channel inactivation. As for the Q1957X mutation, our current hypothesis is that this mutant generates no currents. In addition, we hypothesize that the Q1957X mutant will act as a dominant negative regulator of coexpressed WT channels via the unfolded protein response.

The Silent K+ Channel Subunit, KV6.4. Influences the Gating Charge Movement of KV2.1 in a Heterotetrameric Channel Complex

Voltage-gated K+ (Kv) channels are tetramers of α-subunits that detect changes in membrane potential (V) by a positively charged (Q) voltage-sensing domain (VSD). Molecular movements of VSDs lead to charge displacement that can be recorded as transient gating currents (IQ), which subsequently results in channel gating. The silent Kv subunit, Kv6.4, does not form functional homotetramers; however, it can tetramerize with Kv2.1 subunits to form functional Kv2.1/Kv6.4 heterotetramers, with a proposed 3:1 stoichiometry. Previously we showed that Kv6.4 subunits exert a significant (∼40 mV) hyperpolarizing shift in the voltage-dependent inactivation of heterotetrameric Kv2.1/Kv6.4 channels, as compared to Kv2.1 homotetramers, without significant effects on activation gating. However, the underlying mechanism remains unclear. To address this we analyzed ionic and IQ recordings from heterotetrameric Kv2.1/Kv6.4 channels transiently expressed in HEK293A cells. Half-maximal displacement of gating charge (Q1/2) for Kv2.1 homotetramers was −26 mV, as determined from the charge-voltage (Q-V) curve. Analysis of the decay time constant of IQ-ON as a function of voltage resulted in a bell shaped curve with a maximal time constant around the midpoint potential of −20 mV. Co-expressing Kv6.4 with Kv2.1 resulted in earlier charge movement as evident from a ∼16 mV hyperpolarizing shift in the Q-V curve. Furthermore, we observed a double bell shaped curve for the decay time constant, with maximal time constants around −20 mV and −70 mV; the latter corresponding to the Kv6.4-induced ∼40 mV hyperpolarizing shift in the voltage-dependence of channel inactivation. Therefore, we suggest that this more negatively located ON-gating component presumably reflects the voltage-dependence of the Kv6.4 subunit within the Kv2.1/Kv6.4 heterotetramer, and that the VSD movement of only the Kv6.4 subunit is sufficient to induce closed state channel inactivation.

The gamma subunit runs in the family

Ion channels are ubiquitous membrane proteins that play fundamental roles in cell physiology such as muscle contraction, secretion, excitability and gene expression, among others. Ion channels that are permeant to Ca2+ are especially relevant to cells where this cation acts as a second messenger. In skeletal muscle, the Cav1.1 channel, a key element in excitation–contraction coupling, is composed of four subunits, α1, β, α2δ and γ (Arikkath & Campbell, 2003). The γ subunit was initially described as a major component of the muscle channel complex (Curtis & Catterall, 1984) and while heart muscle and other excitable cells express their α1, β and α2δ subunit counterparts, the γ subunit appeared for many years to be uniquely expressed in skeletal muscle (Jay et al. 1990) and its role on channel function remained largely unknown. This view changed dramatically when it was found that the stargazer mouse, a mutant mouse with epileptic seizures, is deficient in stargazin, a brain protein that is similar to the muscle γ subunit and whose absence leads to alterations in neuronal Ca2+ channel function. When stargazin is expressed in model systems, Ca2+ channel inactivation is accentuated and channel availability is significantly reduced (Letts et al. 1998). In skeletal muscle, inactivation of Cav1.1 channels is shifted to more positive potentials when the γ subunit is absent (Freise et al. 2000). In both cases, the role of the skeletal muscle γ1 subunit and the role of the neuronal γ2 subunit are to avoid an inappropriate Ca2+ entry. To these two family members of γ subunits, other members, γ3, γ4 and γ5, were added using conserved structural criteria (Burgess et al. 1999) and more recently, a cluster of three new γ genes (γ6, γ7 and γ8) was described and it was found that the profile of gene expression of these eight γ subunits is wider than previously thought (Burgess et al. 2001). Soon, new roles for members of the γ family emerged such as trafficking of ligand-gated ion channels to postsynaptic sites (Chu et al. 2001). More recently, a role of γ6 subunit as a potential regulator of the low-threshold cardiac Ca2+ channel Cav3.1 was characterized. It was found that γ6 has the unique ability to decrease current density by more than half without changing the voltage dependence of channel inactivation or the amount of total Cav3.1 protein (Hansen et al. 2004). It is unclear what makes γ6 so unique in the sense that it is the only γ subunit known to modulate the low-voltage-activated Ca2+ channel in atria, as is whether or not this subunit actually forms a physical association with the Cav3.1 channel. If it does, what is the nature of this interaction? In this issue of The Journal of Physiology, Lin et al. (2008) identify a domain in γ6 that is responsible for the inhibition of Cav3.1 currents. Furthermore, they describe a unique GxxxA motif within this domain that is required for the function of the γ6 subunit, pointing to helix–helix interactions and demonstrate physical interactions of this subunit with the channels. The γ6 subunit is highly selective on its inhibitory action, with no effects on the high-voltage-activated Ca2+ channel that is also expressed in heart muscle. This implies a fine modulation of Ca2+ entry in atria. Is this also the case in other tissues? Are there other unsuspected functions for the γ6 subunit? For example, the expression of low-voltage-activated Ca2+ channels in adult skeletal muscle is practically non-existent; it is the high-voltage-activated Cav1.1 channel that is the predominant one, and yet γ6 is expressed in this tissue.

A Single Amino Acid Difference betweenEther-a-go-go-Related Gene Channel Subtypes Determines Differential Sensitivity to a Small Molecule Activator

Activators of human ether-a-go-go-related gene 1 (hERG1) channels, such as (3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243), reverse the effect of hERG1 blockers and shorten the duration of cardiac action potentials. RPR260243 (RPR) slows the rate of deactivation and shifts the voltage dependence of channel inactivation to more positive potentials. We recently mapped the binding site for RPR to several residues located near the cytoplasmic ends of the S5 and S6 helices of the hERG1 subunit. These residues are conserved in the highly homologous ether-a-go-go-related gene 3 (ERG3) subunit; however, RPR blocks ERG3 channels. Here, we compare hERG1 and rat ERG3 (rERG3) channels to explore the molecular basis for differential channel sensitivity to RPR. Channels were heterologously expressed in Xenopus laevis oocytes, and currents were recorded using the two-electrode voltage-clamp technique. Site-directed mutagenesis was used to swap the two residues within the putative binding domain that differed between hERG1 and rERG3. The differential sensitivity of hERG1 and rERG3 channels to the agonist effect of RPR could be accounted for by a single S5 residue (Thr556 in hERG1, Ile558 in rERG3). A Thr in this position favors agonist activity, whereas an Ile reveals a secondary blocking effect of RPR.

The effect of the outermost basic residues in the S4 segments of the CaV3.1 T-type calcium channel on channel gating

The contribution of voltage-sensing S4 segments in domains I to IV of the T-type Ca(V)3.1 calcium channel to channel gating was investigated by the replacement of the uppermost charged arginine residues by neutral cysteines. In each construct, either a single (R180C, R834C, R1379C or R1717C) or a double (two adjacent domains) mutation was introduced. We found that the neutralisation of the uppermost arginines in the IS4, IIS4 and IIIS4 segments shifted the voltage dependence of channel activation in a hyperpolarising direction, with the most prominent effect in the IS4 mutant. In contrast, the voltage dependence of channel inactivation was shifted towards more negative membrane potentials in all four single mutant channels, and these effects were more pronounced than the effects on channel activation. Recovery from inactivation was affected by the IS4 and IIIS4 mutations. In double mutants, the effects on channel inactivation and recovery from inactivation, but not on channel activation, were additive. Exposure of mutant channels to the reducing agent dithiothreitol did not alter channel properties. In summary, our data indicate that the S4 segments in all four domains of the Ca(V)3.1 calcium channels contribute to voltage sensing during channel inactivation, while only the S4 segments in domains I, II and III play such role in channel activation. Furthermore, the removal of the outermost basic amino acids from the IVS4 and IIIS4 and, to a lesser extent, from IS4 segments stabilised the open state of the channel, whereas neutralization from that of IIS4 destabilised it.

Cytoplasmic ATP-sensing Domains Regulate Gating of Skeletal Muscle ClC-1 Chloride Channels

ClC proteins are a family of chloride channels and transporters that are found in a wide variety of prokaryotic and eukaryotic cell types. The mammalian voltage-gated chloride channel ClC-1 is important for controlling the electrical excitability of skeletal muscle. Reduced excitability of muscle cells during metabolic stress can protect cells from metabolic exhaustion and is thought to be a major factor in fatigue. Here we identify a novel mechanism linking excitability to metabolic state by showing that ClC-1 channels are modulated by ATP. The high concentration of ATP in resting muscle effectively inhibits ClC-1 activity by shifting the voltage gating to more positive potentials. ADP and AMP had similar effects to ATP, but IMP had no effect, indicating that the inhibition of ClC-1 would only be relieved under anaerobic conditions such as intense muscle activity or ischemia, when depleted ATP accumulates as IMP. The resulting increase in ClC-1 activity under these conditions would reduce muscle excitability, thus contributing to fatigue. We show further that the modulation by ATP is mediated by cystathionine beta-synthase-related domains in the cytoplasmic C terminus of ClC-1. This defines a function for these domains as gating-modulatory domains sensitive to intracellular ligands, such as nucleotides, a function that is likely to be conserved in other ClC proteins.

Modulation of the Cardiac Sodium Channel Nav1.5 by Fibroblast Growth Factor Homologous Factor 1B

We have previously shown that fibroblast growth factor homologous factor 1B (FHF1B), a cytosolic member of the fibroblast growth factor family, associates with the sensory neuron-specific channel Na(v)1.9 but not with the other sodium channels present in adult rat dorsal root ganglia neurons. We show in this study that FHF1B binds to the C terminus of the cardiac voltage-gated sodium channel Na(v)1.5 and modulates the properties of the channel. The N-terminal 41 amino acid residues of FHF1B are essential for binding to Na(v)1.5, and the conserved acidic rich domain (amino acids 1773-1832) in the C terminus of Na(v)1.5 is sufficient for association with this factor. Binding of the growth factor to recombinant wild type human Na(v)1.5 in human embryonic kidney 293 cells produces a significant hyperpolarizing shift in the voltage dependence of channel inactivation. An aspartic acid to glycine substitution at position 1790 of the channel, which underlies one of the LQT-3 phenotypes of cardiac arrythmias, abolishes the interaction of the Na(v)1.5 channel with FHF1B. This is the first report showing that interaction with a growth factor can modulate properties of a voltage-gated sodium channel.

Human ether-à-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors.

Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.

CLINICAL CONCENTRATIONS OF ISOFLURANE SIGNIFICANTLY SUPPRESSES SODIUM CURRENTS IN RAT DORSAL ROOT GANGLIA

S519 Introduction: Over the last decade a number of studies have revealed that clinical concentrations of volatile anesthetics alter the discharge pattern and action potential properties of mammalian CNS and peripheral neurons [1]. In thin and unmyelinated neurons, which represent a substantial fraction of intracortical fibers and sensory afferents, anesthetics also have been found to alter signal propagation. [1] These neuronal functions all depend on the proper function of voltage-dependent sodium channels, making these integral membrane proteins potentially important molecular targets of general anesthetics. Although early studies examining anesthetic modification of sodium channel currents in non-mammalian preparations found them to be relatively insensitive targets compared with ligand-gated channels, a recent report has found that rat brain-Ila sodium channels expressed in a mammalian cell line are as sensitive as GABA-A receptors to clinical concentrations of volatile anesthetics [2]. It therefore has become essential to determine whether sodium channels in mammalian neurons have a similar anesthetic sensitivity as the expressed channels. To examine this question, we examined isoflurane modification of sodium currents in cultured rat Dorsal Root Ganglia (DRG). Methods: Freshly dissociated and primary cultured neurons of DRG from 5-30 day rats were obtained after enzymatic digestion with collagenase and/or trypsin. Sodium currents were measured using the whole cell patch clamp configuration; the extracellular Hanks' balanced salt solution additionally contained 5 mM HEPES and 0.1 mM CdCl2, pH 7.2, while the internal solution was 140 mM CsF, 10 mM NaCl, 5 mM HEPES, pH 7.4. Control measurements of the cells elicited normal action potentials and ionic currents. Sodium currents and anesthetic suppression were measured with depolarizing test potentials from a hyperpolarizing holding potential. Aqueous concentrations of isoflurane, which are relatively independent of experimental temperature [3], were measured by gas chromatography. All experiments were conducted at room temperature (22-25[degree sign]C). Results: Similar to results with the rat brain Ila sodium channels, isoflurane suppressed sodium currents through at least two mechanisms: a) a voltage-independent suppression of resting or open channels at hyperpolarized potentials, and b) a hyperpolarizing shift in the voltage-dependence of channel inactivation. Unlike the expressed channels, however, even high concentrations of isoflurane could not completely block sodium currents at potentials more negative than -80 mV. At -80 mV, isoflurane maximally suppressed 54 +/- 7.0% of sodium current (n=11, concentrations up to 5.4 mM isoflurane). The range of maximal suppression was 40-75%. Averaging data from the 11 cells and fitting to a rectangular hyperbola yielded a maximal suppression of 0.49 with an ED50=0.17 mM, slightly lower than aqueous MAC concentrations. The midpoint of sodium channel inactivation was about -50 mV. Similar to rat brain sodium channels, 0.54 mM isoflurane (twice MAC) shifted the inactivation midpoint by about -20 mV, resulting in significantly greater current suppression at these potentials (about 80% block at -60mV, compared with about 20% at -80 mV). Conclusions: Clinical concentrations of isoflurane caused a large depolarizing shift in steady-state channel inactivation, resulting in the block of a significant fraction (>50%) of sodium currents in DRG neurons at resting membrane potentials. These results support the hypothesis that some mammalian neuronal sodium channels are sensitive to clinical concentrations of volatile anesthetics in situ, and that their anesthetic modification may directly alter neuronal function.

Developmental cardiac electrophysiology recent advances in cellular physiology

This overview of cardiac ion channel development does not suggest any particular theme underlying the expression or regulation of all channel subtypes. Calcium and potassium channels generally exhibit increased expression in more mature hearts. However, this increase in channel number or activity as determined under voltage clamp conditions may not be translated into increased activity in vivo. Concomitant changes in other physiological factors such as local intracellular Ca2+ accumulation, increased resting membrane potential and decreased heart rate in mature heart may inhibit or augment channel activity. Na(+)-Ca2+ exchange activity appears to decrease with development, possibly reflecting its decreasing role in both systolic and diastolic Ca2+ regulation. Na+ channel activity follows a middle course, exhibiting little change in channel conductance. The reported shift in the voltage dependence of channel inactivation toward more negative membrane potentials may reflect a concomitant shift in the resting membrane potential in mature heart. However, this change is in the direction opposite to that reported for L-type Ca2+ channel inactivation, suggesting that the regulation of these channels is not modulated by a common factor such as membrane surface charge. A detailed characterization of multiple channel subtypes in mature myocardium has resulted in significant advances in models of the cardiac action potential and excitation-contraction coupling. Recently, developmental changes in ion channel physiology have been described, setting the stage for a comparable elucidation of the ontogeny of the cardiac action potential. Ca2+ and K+ channel currents generally become more prominent with development. In contrast, developmental changes in Na+ currents are less dramatic and Na(+)-Ca2+ exchange currents appear to decrease with age. These changes may, in part, be reflected by the increasingly important role of transsarcolemmal Ca2+ influx in triggering Ca2+ release from the SR in mature heart as compared to its direct role of providing Ca2+ for cell contraction in immature heart. These developmental changes in ion channel expression and function are likely to have a significant effect on the generation of the action potential in individual myocytes. Developmental changes in the characteristics of the action potential may then have a major influence on the initiation, propagation and termination of autonomic, triggered, and re-entrant arrhythmias. Progress in this area provides an essential foundation for the evolution of a systematic approach to pediatric arrhythmias comparable to that under development for mature heart [3].