Modulation of synaptic potentials and cell excitability by dendritic K<Subscript>IR</Subscript> and K<Subscript>As</Subscript> channels in nucleus accumbens medium spiny neurons: A computational study

The nucleus accumbens (NAc), a critical structure of the brain reward circuit, is implicated in normal goal-directed behaviour and learning as well as pathological conditions like schizophrenia and addiction. Its major cellular substrates, the medium spiny (MS) neurons, possess a wide variety of dendritic active conductances that may modulate the excitatory post synaptic potentials (EPSPs) and cell excitability. We examine this issue using a biophysically detailed 189-compartment stylized model of the NAc MS neuron, incorporating all the known active conductances. We find that, of all the active channels, inward rectifying K⁺ (K_IR) channels play the primary role in modulating the resting membrane potential (RMP) and EPSPs in the down-state of the neuron. Reduction in the conductance of K_IR channels evokes facilitatory effects on EPSPs accompanied by rises in local input resistance and membrane time constant. At depolarized membrane potentials closer to up-state levels, the slowly inactivating A-type potassium channel (K_As) conductance also plays a strong role in determining synaptic potential parameters and cell excitability. We discuss the implications of our results for the regulation of accumbal MS neuron biophysics and synaptic integration by intrinsic factors and extrinsic agents such as dopamine.     

The h-Current in the Substantia Nigra pars Compacta Neurons: A Re-examination

The properties of the hyperpolarization-activated cation current (I_h) were investigated in rat substantia nigra - pars compacta (SNc) principal neurons using patch-clamp recordings in thin slices. A reliable identification of single dopaminergic neurons was made possible by the use of a transgenic line of mice expressing eGFP under the tyrosine hydroxylase promoter. The effects of temperature and different protocols on the I_h kinetics showed that, at 37°C and minimizing the disturbance of the intracellular milieu with perforated patch, this current actually activates at potentials more positive than what is generally indicated, with a half-activation potential of −77.05 mV and with a significant level of opening already at rest, thereby substantially contributing to the control of membrane potential, and ultimately playing a relevant function in the regulation of the cell excitability. The implications of the known influence of intracellular cAMP levels on I_h amplitude and kinetics were examined. The direct application of neurotransmitters (DA, 5-HT and noradrenaline) physiologically released onto SNc neurons and known to act on metabotropic receptors coupled to the cAMP pathway modify the I_h amplitude. Here, we show that direct activation of dopaminergic and of 5-HT receptors results in I_h inhibition of SNc DA cells, whereas noradrenaline has the opposite effect. Together, these data suggest that the modulation of I_h by endogenously released neurotransmitters acting on metabotropic receptors –mainly but not exclusively linked to the cAMP pathway- could contribute significantly to the control of SNc neuron excitability.     

Contributions of <Emphasis Type="Italic">I</Emphasis><Subscript>h</Subscript> to feature selectivity in layer II stellate cells of the entorhinal cortex

Stellate cells (SCs) of the entorhinal cortex generate prominent subthreshold oscillations that are believed to be important contributors to the hippocampal theta rhythm. The slow inward rectifier I _h is expressed prominently in SCs and has been suggested to be a dominant factor in their integrative properties. We studied the input-output relationships in stellate cells (SCs) of the entorhinal cortex, both in control conditions and in the presence of the I _h antagonist ZD7288. Our results show that I _h is responsible for SCs’ subthreshold resonance, and contributes to enhanced spiking reliability to theta-rich stimuli. However, SCs still exhibit other traits of rhythmicity, such as subthreshold oscillations, under I _h blockade. To clarify the effects of I _h on SC spiking, we used a generalized form of principal component analysis to show that SCs select particular features with relevant temporal signatures from stimuli. The spike-selected mix of those features varies with the frequency content of the stimulus, emphasizing the inherent nonlinearity of SC responses. A number of controls confirmed that this selectivity represents a stimulus-induced change in the cellular input-output relationship rather than an artifact of the analysis technique. Sensitivity to slow features remained statistically significant in ZD7288. However, with I _h blocked, slow stimulus features were less predictive of spikes and spikes conveyed less information about the stimulus over long time scales. Together, these results suggest that I _h is an important contributor to the input-output relationships expressed by SCs, but that other factors in SCs also contribute to subthreshold oscillations and nonlinear selectivity to slow features. Action Editor: Xiao-Jing Wang     

The h-Current in Periglomerular Dopaminergic Neurons of the Mouse Olfactory Bulb

The properties of the hyperpolarization-activated cation current (I_h) were investigated in rat periglomerular dopaminergic neurons using patch-clamp recordings in thin slices. A reliable identification of single dopaminergic neurons was made possible by use of a transgenic line of mice expressing eGFP under the tyrosine hydroxylase promoter. At 37 °C and minimizing the disturbance of the intracellular milieu with perforated patches, this current shows a midpoint of activation around −82.7 mV, with a significant level of opening already at rest, thereby giving a substantial contribution to the resting potential, and ultimately playing a relevant function in the control of the cell excitability. The blockage of I_h has a profound influence on the spontaneous firing of these neurons, which result as strongly depressed. However the effect is not due to a direct role of the current in the pacemaker process, but to the I_h influence on the resting membrane potential. I_h kinetics is sensitive to the intracellular levels of cAMP, whose increase promotes a shift of the activation curve towards more positive potentials. The direct application of DA and 5-HT neurotransmitters, physiologically released onto bulbar dopaminergic neurons and known to act on metabotropic receptors coupled to the cAMP pathway, do not modifythe I_h amplitude. On the contrary, noradrenaline almost halves the I_h amplitude. Our data indicate that the HCN channels do not participate directly to the pacemaker activity of periglomerular dopaminergic neurons, but influence their resting membrane potential by controlling the excitability profile of these cells, and possibly affecting the processing of sensory information taking place at the entry of the bulbar circuitry.     

The vicissitudes of the pacemaker current <Emphasis Type="Italic">I</Emphasis><Subscript>Kdd</Subscript> of cardiac purkinje fibers

Summary The mechanisms underlying the pacemaker current in cardiac tissues is not agreed upon. The pacemaker potential in Purkinje fibers has been attributed to the decay of the potassium current I _Kdd. An alternative proposal is that the hyperpolarization-activated current I _f underlies the pacemaker potential in all cardiac pacemakers. The aim of this review is to retrace the experimental development related to the pacemaker mechanism in Purkinje fibers with reference to findings about the pacemaker mechanism in the SAN as warranted. Experimental data and their interpretation are critically reviewed. Major findings were attributed to K⁺ depletion in narrow extracellular spaces which would result in a time dependent decay of the inward rectifier current I _K1. In turn, this decay would be responsible for a “fake” reversal of the pacemaker current. In order to avoid such a postulated depletion, Ba²⁺ was used to block the decay of I _K1. In the presence of Ba²⁺ the time-dependent current no longer reversed and instead increased with time and more so at potentials as negative as −120 mV. In this regard, the distinct possibility needs to be considered that Ba²⁺ had blocked I _Kdd (and not only I _K1). That indeed this was the case was demonstrated by studying single Purkinje cells in the absence and in the presence of Ba²⁺. In the absence of Ba²⁺, I _Kdd was present in the pacemaker potential range and reversed at E _K. In the presence of Ba²⁺, I _Kdd was blocked and I _f appeared at potentials negative to the pacemaker range. The pacemaker potential behaves in a manner consistent with the underlying I _Kdd but not with I _f. The fact that I _f is activated on hyperpolarization at potential negative to the pacemaker range makes it suitable as a safety factor to prevent the inhibitory action of more negative potentials on pacemaker discharge. It is concluded that the large body of evidence reviewed proves the pacemaker role of I _Kdd (but not of I _f) in Purkinje fibers.     

Computational study of enhanced excitability in Hermissenda: membrane conductances modulated by 5-HT

Serotonin (5-HT) applied to the exposed but otherwise intact nervous system results in enhanced excitability of Hermissenda type-B photoreceptors. Several ion currents in the type-B photoreceptors are modulated by 5-HT, including the A-type K⁺ current (I_K,A), sustained Ca²⁺ current (I_Ca,S), Ca-dependent K⁺ current (I_K,Ca), and a hyperpolarization-activated inward rectifier current (I_h). In this study, we developed a computational model that reproduces physiological characteristics of type B photoreceptors, e.g. resting membrane potential, dark-adapted spike activity, spike width, and the amplitude difference between somatic and axonal spikes. We then used the model to investigate the contribution of different ion currents modulated by 5-HT to the magnitudes of enhanced excitability produced by 5-HT. Ion currents were systematically varied within limits observed experimentally, both individually and in combinations. A reduction of I_K,A or I_K,Ca, or an increase in I_h enhanced excitability by 20–50%. Decreasing I_Ca,S produced a dramatic decrease in excitability. Reductions of I_K,V produced only minimal increases in excitability, suggesting that I_K,V probably plays a minor role in 5-HT induced enhanced excitability. Combinations of changes in I_K,A, I_K,Ca, I_h and I_Ca,S produced increases in excitability comparable to experimental observations. After 5-HT application, the cell's depolarization force is shifted from the I_h–I_Ca,S combination to predominantly I_h.     

Targeted Deletion of Kcne2 Impairs HCN Channel Function in Mouse Thalamocortical Circuits

Background

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the pacemaking current, I_h, which regulates neuronal excitability, burst firing activity, rhythmogenesis, and synaptic integration. The physiological consequence of HCN activation depends on regulation of channel gating by endogenous modulators and stabilization of the channel complex formed by principal and ancillary subunits. KCNE2 is a voltage-gated potassium channel ancillary subunit that also regulates heterologously expressed HCN channels; whether KCNE2 regulates neuronal HCN channel function is unknown.

Methodology/Principal Findings

We investigated the effects of Kcne2 gene deletion on I_h properties and excitability in ventrobasal (VB) and cortical layer 6 pyramidal neurons using brain slices prepared from Kcne2 ^+/+ and Kcne2 ^−/− mice. Kcne2 deletion shifted the voltage-dependence of I_h activation to more hyperpolarized potentials, slowed gating kinetics, and decreased I_h density. Kcne2 deletion was associated with a reduction in whole-brain expression of both HCN1 and HCN2 (but not HCN4), although co-immunoprecipitation from whole-brain lysates failed to detect interaction of KCNE2 with HCN1 or 2. Kcne2 deletion also increased input resistance and temporal summation of subthreshold voltage responses; this increased intrinsic excitability enhanced burst firing in response to 4-aminopyridine. Burst duration increased in corticothalamic, but not thalamocortical, neurons, suggesting enhanced cortical excitatory input to the thalamus; such augmented excitability did not result from changes in glutamate release machinery since miniature EPSC frequency was unaltered in Kcne2 ^−/− neurons.

Conclusions/Significance

Loss of KCNE2 leads to downregulation of HCN channel function associated with increased excitability in neurons in the cortico-thalamo-cortical loop. Such findings further our understanding of the normal physiology of brain circuitry critically involved in cognition and have implications for our understanding of various disorders of consciousness.     

Hyperpolarization-Activated Current (Ih) Is Reduced in Hippocampal Neurons from Gabra5?/? Mice

Changes in the expression of γ-aminobutyric acid type A (GABA_A) receptors can either drive or mediate homeostatic alterations in neuronal excitability. A homeostatic relationship between α5 subunit-containing GABA_A (α5GABA_A) receptors that generate a tonic inhibitory conductance, and HCN channels that generate a hyperpolarization-activated cation current (I_h) was recently described for cortical neurons, where a reduction in I_h was accompanied by a reciprocal increase in the expression of α5GABA_A receptors resulting in the preservation of dendritosomatic synaptic function. Here, we report that in mice that lack the α5 subunit gene (Gabra5−/−), cultured embryonic hippocampal pyramidal neurons and ex vivo CA1 hippocampal neurons unexpectedly exhibited a decrease in I_h current density (by 40% and 28%, respectively), compared with neurons from wild-type (WT) mice. The resting membrane potential and membrane hyperpolarization induced by blockade of I_h with ZD-7288 were similar in cultured WT and Gabra5−/− neurons. In contrast, membrane hyperpolarization measured after a train of action potentials was lower in Gabra5−/− neurons than in WT neurons. Also, membrane impedance measured in response to low frequency stimulation was greater in cultured Gabra5−/− neurons. Finally, the expression of HCN1 protein that generates I_h was reduced by 41% in the hippocampus of Gabra5−/− mice. These data indicate that loss of a tonic GABAergic inhibitory conductance was followed by a compensatory reduction in I_h. The results further suggest that the maintenance of resting membrane potential is preferentially maintained in mature and immature hippocampal neurons through the homeostatic co-regulation of structurally and biophysically distinct cation and anion channels.     

Remodeling of Hyperpolarization-Activated Current,Ih, in Ah-Type Visceral Ganglion Neurons Following Ovariectomy in Adult Rats

Hyperpolarization-activated currents (I_h) mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels modulate excitability of myelinated A− and Ah-type visceral ganglion neurons (VGN). Whether alterations in I_h underlie the previously reported reduction of excitability of myelinated Ah-type VGNs following ovariectomy (OVX) has remained unclear. Here we used the intact nodose ganglion preparation in conjunction with electrophysiological approaches to examine the role of I_h remodeling in altering Ah-type neuron excitability following ovariectomy in adult rats. Ah-type neurons were identified based on their afferent conduction velocity. Ah-type neurons in nodose ganglia from non-OVX rats exhibited a voltage ‘sag’ as well as ‘rebound’ action potentials immediately following hyperpolarizing current injections, which both were suppressed by the I_h blocker ZD7288. Repetitive spike activity induced afterhyperpolarizations lasting several hundreds of milliseconds (termed post-excitatory membrane hyperpolarizations, PEMHs), which were significantly reduced by ZD7288, suggesting that they resulted from transient deactivation of I_h during the preceding spike trains. Ovariectomy reduced whole-cell I_h density, caused a hyperpolarizing shift of the voltage-dependence of I_h activation, and slowed I_h activation. OVX-induced I_h remodeling was accompanied by a flattening of the stimulus frequency/response curve and loss of PEMHs. Also, HCN1 mRNA levels were reduced by ∼30% in nodose ganglia from OVX rats compared with their non-OVX counterparts. Acute exposure of nodose ganglia to 17beta-estradiol partly restored I_h density and accelerated I_h activation in Ah-type cells. In conclusion, I_h plays a significant role in modulating the excitability of myelinated Ah-type VGNs in adult female rats.     

Transient K<Superscript>+</Superscript> Current is Blocked by Lanthanum in <Emphasis Type="Italic">Drosophila</Emphasis> Neurons

The potassium A-current (IK_A) is important in regulating the membrane potential between action potentials. The whole-cell patch-clamp technique was applied to cultured Drosophila neurons derived from embryonic neuroblasts. IK_A was measured from neurons before and after application of 0.1 mM lanthanum to the external saline. IK_A was smaller in the lanthanum-containing saline (7±1 pA) than in the control saline (34±6 pA). Activation and inactivation of IK_A were unchanged by lanthanum. These results suggest that lanthanum neurotoxicity may lead to increased neuronal excitability. Moreover, given this inhibition of IK_A, lanthanum should not be used to block calcium current in studies of K⁺ currents.     

Inhibition modifies the effects of slow calcium-activated potassium channels on epileptiform activity in a neuronal network model

Generation of epileptiform activity typically results from a change in the balance between network excitation and inhibition. Experimental evidence indicates that alterations of either synaptic activity or intrinsic membrane properties can produce increased network excitation. The slow Ca²⁺-activated K⁺ currents (sI_AHP) are important modulators of neuronal firing rate and excitability and have important established and potential roles in epileptogenesis. While the effects of changes in sI_AHP on individual neuronal excitability are readily studied and well established, the effects of such changes on network behavior are less well known. The experiments here utilize a defined small network model of multicompartment pyramidal cells and an inhibitory interneuron to study the effects of changes in sI_AHP on network behavior. The benefits of this model system include the ability to observe activity in all cells in a network and the effects of interactions of multiple simultaneous influences. In the model with no inhibitory interneuron, increasing sI_AHP results in progressively decreasing burst activity. Adding an inhibitory interneuron changes the observed effects; at modest inhibitory strengths, increasing sI_AHP in all network neurons actually results in increased network bursting (except at very high values). The duration of the burst activity is influenced by the length of delay in a feedback loop, with longer loops resulting in more prolonged bursting. These observations illustrate that the study of potential antiepileptogenic membrane effects must be extended to realistic networks. Network inhibition can dramatically alter the observations seen in pure excitatory networks.     

The Cdk5 Kinase Downregulates ATP-Gated Ionotropic P2X<Subscript>3</Subscript> Receptor Function Via Serine Phosphorylation

Cdk5 is an endogenous kinase activated by the neuronal-specific protein p35 and implicated in multiple neuronal functions, including modulation of certain pain responses. We investigated whether Cdk5 could regulate ATP-gated P2X₃ receptors that are members of the family of membrane proteins expressed by sensory neurons to transduce nociception in baseline and chronic pain. To study the potential P2X₃ receptor modulation by Cdk5, we co-transfected rat P2X₃ receptors and Cdk5 into HEK cells and observed increased P2X₃ receptor serine phosphorylation together with downregulation of receptor currents only when these genes were transfected together with the gene of the Cdk5 activator p35. The changes in receptor responses were limited to depressed current amplitude as desensitization and recovery were not altered. Transfection of p35 with P2X₃ similarly downregulated receptor responses, suggesting that this phenomenon could be observed even with constitutive Cdk5. The present data indicate a novel target to express the action of Cdk5 on membrane proteins involved in pain perception.     

Prostaglandin E<Subscript>2</Subscript> induces upregulation of Na<Superscript>+</Superscript> transport across<Emphasis Type="Italic"> Xenopus</Emphasis> lung epithelium

The apical mucus on pulmonary epithelia is not only critical for physiological functions such as gas exchange or inflammatory processes, but also contains surfactants and multiple molecules that mediate cellular responses. A tight control of transepithelial ion transport maintains viscosity of this layer and, e.g., the amiloride-sensitive sodium channels (ENaCs) in lung epithelia of vertebrates are the most important regulatory sites for transcellular sodium uptake. Dysfunction of this sodium transport results in reduced liquid absorption and causes massive problems with gas exchange. We used dissected lungs of Xenopus laevis in Ussing chambers to investigate the influence of prostaglandin E₂ (PGE₂) on the regulation of short-circuit current (I _SC) and amiloride-sensitive sodium absorption (I _ami). Apical application of PGE₂ (1 M) increased I _SC by 38% and I _ami by approximately 60%. In contrast, a different prostaglandin, PGI₂, neither affected I _SC nor I _ami. Forskolin increased current to a similar magnitude and preincubation of the lung with an RP-isomer of cyclic AMP, an inhibitor of proteinkinase A (PKA), abolished the effects of both PGE₂ and forskolin. Transepithelial Na⁺ uptake was also upregulated by the prostaglandin receptor agonists misoprostol and sulprostone . The I _ami in Xenopus oocytes that heterologously expressed ENaCs was not affected by PGE₂.Abbreviations ACTH adrenocorticotropic hormone - ENaC epithelial sodium channel - hENaC epithelial sodium channels from human lung - ORI oocyte Ringers solution - PKA protein kinase A - R _T transepithelial resistance - V _T transepithelial potential - xENaC epithelial sodium channels from Xenopus nephron - I _ami amiloride-sensitive current - I _SC short-circuit current - NRS normal Ringers solution - PGE ₂ prostaglandin E₂ Communicated by G. Heldmaier     

HCN4 knockdown in dorsal hippocampus promotes anxiety‐like behavior in mice

Hyperpolarization‐activated and cyclic nucleotide‐gated (HCN) channels mediate the I_h current in the murine hippocampus. Disruption of the I_h current by knockout of HCN1, HCN2 or tetratricopeptide repeat‐containing Rab8b‐interacting protein has been shown to affect physiological processes such as synaptic integration and maintenance of resting membrane potentials as well as several behaviors in mice, including depressive‐like and anxiety‐like behaviors. However, the potential involvement of the HCN4 isoform in these processes is unknown. Here, we assessed the contribution of the HCN4 isoform to neuronal processing and hippocampus‐based behaviors in mice. We show that HCN4 is expressed in various regions of the hippocampus, with distinct expression patterns that partially overlapped with other HCN isoforms. For behavioral analysis, we specifically modulated HCN4 expression by injecting recombinant adeno‐associated viral (rAAV) vectors mediating expression of short hairpin RNA against hcn4 (shHcn4) into the dorsal hippocampus of mice. HCN4 knockdown produced no effect on contextual fear conditioning or spatial memory. However, a pronounced anxiogenic effect was evident in mice treated with shHcn4 compared to control littermates. Our findings suggest that HCN4 specifically contributes to anxiety‐like behaviors in mice.     

Mutant SOD1 protein increases Na<Subscript>v</Subscript>1.3 channel excitability

Amyotrophic lateral sclerosis (ALS) is a lethal paralytic disease caused by the degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) are present in ~20% of familial ALS and ~2% of all ALS cases. The most common SOD1 gene mutation in North America is a missense mutation substituting valine for alanine (A4V). In this study, we analyze sodium channel currents in oocytes expressing either wild-type or mutant (A4V) SOD1 protein. We demonstrate that the A4V mutation confers a propensity to hyperexcitability on a voltage-dependent sodium channel (Na_v1.3) mediated by heightened total Na⁺ conductance and a hyperpolarizing shift in the voltage dependence of Na_v1.3 activation. To estimate the impact of these channel effects on excitability in an intact neuron, we simulated these changes in the program NEURON; this shows that the changes induced by mutant SOD1 increase the spontaneous firing frequency of the simulated neuron. These findings are consistent with the view that excessive excitability of neurons is one component in the pathogenesis of this disease.     

PTX-induced hyperexcitability affects dendritic shape and GABAergic synapse density but not synapse distribution during <Emphasis Type="Italic">Manduca</Emphasis> postembryonic motoneuron development

During the metamorphosis of the holometabolous insect, Manduca sexta, the postembryonic acquisition of adult specific motor behaviors is accompanied by changes in dendritic architecture, membrane currents, and input synapses of identified motoneurons. This study aims to test whether increased activity affects dendritic architecture and sub-dendritic input synapse distribution of the identified flight motoneuron 5 (MN5). Systemic injections of the chloride channel blocker, picrotoxin (PTX), during early pupal stages increase pupal reflex responsiveness, but overall development is not impaired. MN5 input resistance, resting membrane potential, and spiking threshold are not affected. Bath application of PTX to isolated ventral nerve cords evokes spiking in pupal and adult flight motoneurons. Quantitative three-dimensional reconstructions of the dendritic tree of the adult MN5 show that systemic PTX injections into early pupae cause dendritic overgrowth and reduce the density of GABAergic inputs. In contrast, the distribution patterns of GABAergic terminals throughout the dendritic tree remain unaltered. This indicates that increased overall excitability might cause dendritic overgrowth and decreased inhibitory input during postembryonic motoneuron remodeling, whereas sub-dendritic synapse targeting might be controlled by activity-independent signals. Behavioral testing reveals that these neuronal changes do not impede the animal’s ability to fly, but impair maximum flight performance.     

Modulation by Clamping: Kv4 and KChIP Interactions

The rapidly inactivating (A-type) potassium channels regulate membrane excitability that defines the fundamental mechanism of neuronal functions such as pain signaling. Cytosolic Kv channel-interacting proteins KChIPs that belong to neuronal calcium sensor (NCS) family of calcium binding EF-hand proteins co-assemble with Kv4 (Shal) α subunits to form a native complex that encodes major components of neuronal somatodendritic A-type K⁺ current, I_SA, in neurons and transient outward current, I_TO, in cardiac myocytes. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Here, I attempt to emphasize the interaction between KChIPs and Kv4 based on recent progress made in understanding the structure complex in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner. Greater insights into molecular mechanism between KChIPs and Kv4 interaction may provide therapeutic potentials of designing compounds aimed at disrupting the protein–protein interaction for treatment of membrane excitability-related disorders. Special issue article in honor of Dr. Ji-Sheng Han.     

Mechanisms of Na<Superscript>+</Superscript>-Ca<Superscript>2+</Superscript> Exchange Inhibition by Amphiphiles in CardiacMyocytes: Importance of Transbilayer Movement

The membrane lipid environment and lipid signaling pathways are potentially involved in the modulation of the activity of the cardiac Na⁺-Ca²⁺ exchanger (NCX). In the present study biophysical mechanisms of interactions of amphiphiles with the NCX and the functional consequences were examined. For this purpose, intracellular Ca²⁺ concentration jumps were generated by laser-flash photolysis of caged Ca²⁺ in guinea-pig ventricular myocytes and Na⁺-Ca²⁺ exchange currents (I_Na/Ca) were recorded in the whole-cell configuration of the patch-clamp technique. The inhibitory effect of amphiphiles increased with the length of the aliphatic chain between C₇ and C₁₀ and was more potent with cationic or anionic head groups than with uncharged head groups. Long-chain cationic amines (C₁₂) exhibited a cut-off in their efficacy in I_Na/Ca inhibition. Analysis of the time-course, comparison with the Ni²⁺-induced I_Na/Ca block and confocal laser scanning microscopy experiments with fluorescent lipid analogs (C₆- and C₁₂-NBD-labeled analogs) suggested that amphiphiles need to be incorporated into the membrane. Furthermore, NCX block appears to require transbilayer movement of the amphiphile to the inner leaflet (“flip”). We conclude that both, hydrophobic and electrostatic interactions between the lipids and the NCX may be important factors for the modulation by lipids and could be relevant in cardiac diseases where the lipid metabolism is altered.This revised version was published online in August 2005 with a corrected cover date.     

The antiproliferative role of ERG K<Superscript>+</Superscript> channels in rat osteoblastic cells

We report on the role of K⁺ currents in the mechanisms regulating the proliferation of UMR 106-01 osteoblastic osteosarcoma cells. Electrophysiological analysis showed that UMR 106-01 cells produce robust K⁺ currents that can be pharmacologically separated into two major components: a E-4031-susceptible current, I _E-4031, and a tetraethylammonium (TEA)-susceptible component, I _TEA. Western blot and RT-PCR analysis showed that I _E-4031 is produced by ether a go-go (eag)-related channels (ERG). Incubation of the cells with E-4031 enhanced their proliferation by 80%. Application of E-4031 in the bath solution did not induce instantaneous changes in the membrane resting potential or in the level of cytosolic calcium; however, the cells were slightly depolarized and the calcium content was significantly increased upon prolonged incubation with the compound. Taken together these findings indicate that ERG channels can impair cell proliferation. This is a novel finding that underscores new modes of regulation of mitosis by voltage-gated K⁺ channels and provides an unexpected insight into the current view of the mechanisms governing bone tissue proliferation.