Ca Currents Research Articles

Regionally specific expression of high-voltage-activated calcium channels in thalamic nuclei of epileptic and non-epileptic rats.

The polygenic origin of generalized absence epilepsy results in dysfunction of ion channels that allows the switch from physiological asynchronous to pathophysiological highly synchronous network activity. Evidence from rat and mouse models of absence epilepsy indicates that altered Ca(2+) channel activity contributes to cellular and network alterations that lead to seizure activity. Under physiological circumstances, high voltage-activated (HVA) Ca(2+) channels are important in determining the thalamic firing profile. Here, we investigated a possible contribution of HVA channels to the epileptic phenotype using a rodent genetic model of absence epilepsy. In this study, HVA Ca(2+) currents were recorded from neurons of three different thalamic nuclei that are involved in both sensory signal transmission and rhythmic-synchronized activity during epileptic spike-and-wave discharges (SWD), namely the dorsal part of the lateral geniculate nucleus (dLGN), the ventrobasal thalamic complex (VB) and the reticular thalamic nucleus (NRT) of epileptic Wistar Albino Glaxo rats from Rijswijk (WAG/Rij) and non-epileptic August Copenhagen Irish (ACI) rats. HVA Ca(2+) current densities in dLGN neurons were significantly increased in epileptic rats compared with non-epileptic controls while other thalamic regions revealed no differences between the strains. Application of specific channel blockers revealed that the increased current was carried by L-type Ca(2+) channels. Electrophysiological evidence of increased L-type current correlated with up-regulated mRNA and protein expression of a particular L-type channel, namely Cav1.3, in dLGN of epileptic rats. No significant changes were found for other HVA Ca(2+) channels. Moreover, pharmacological inactivation of L-type Ca(2+) channels results in altered firing profiles of thalamocortical relay (TC) neurons from non-epileptic rather than from epileptic rats. While HVA Ca(2+) channels influence tonic and burst firing in ACI and WAG/Rij differently, it is discussed that increased Cav1.3 expression may indirectly contribute to increased robustness of burst firing and thereby the epileptic phenotype of absence epilepsy.

Patch-clamp measurement of ICRAC and ORAI channel activity.

Depletion of internal Ca(2+) stores activates store-operated Ca(2+) channels. The most prominent members of this class of channels are Ca(2+) release-activated Ca(2+) (CRAC) channels, which are present in a variety of cell types including immune cells. CRAC channels are composed of ORAI proteins, which are activated by endoplasmic reticulum-bound STIM proteins on Ca(2+) store depletion. The underlying Ca(2+) current is called ICRAC, which is required for many cellular functions including T-cell activation, mast cell activation, Ca(2+)-dependent gene expression, and refilling of internal Ca(2+) stores. To analyze ICRAC or the Ca(2+) current through heterologously expressed ORAI channels, whole-cell patch clamp is the technique of choice. It allows the direct analysis of ion currents through CRAC/ORAI channels. The patch-clamp technique has been used to determine selectivity, permeability, rectification, inactivation, and several other biophysical and pharmacological properties of the channels, and is the most direct and reliable technique to analyze ICRAC.

Selective modulation of cellular voltage-dependent calcium channels by hyperbaric pressure-a suggested HPNS partial mechanism.

Professional deep sea divers experience motor and cognitive impairment, known as High Pressure Neurological Syndrome (HPNS), when exposed to pressures of 100 msw (1.1 MPa) and above, considered to be the result of synaptic transmission alteration. Previous studies have indicated modulation of presynaptic Ca2+ currents at high pressure. We directly measured for the first time pressure effects on the currents of voltage dependent Ca2+ channels (VDCCs) expressed in Xenopus oocytes. Pressure selectivity augmented the current in CaV1.2 and depressed it in CaV3.2 channels. Pressure application also affected the channels' kinetics, such as ƮRise, ƮDecay. Pressure modulation of VDCCs seems to play an important role in generation of HPNS signs and symptoms.

Voltage-gated Ca2+ channels in accessory lobe neurons of the chick

Birds have ten pairs of protrusions, "accessory lobes", on the lateral sides of the lumbosacral spinal cord. It has been proposed that accessory lobes act as a sensory organ of equilibrium and neurons in accessory lobes transmit sensory information to the motor center. We have reported that cells in chick accessory lobes express functional voltage-gated Na(+) and K(+) channels and generate action potentials. In this study, we examined properties of voltage-gated Ca(2+) channels (VGCCs). The amplitude of voltage-gated Ca(2+) channel currents carried by Ca(2+) and Ba(2+) increased gradually during 10 min rather than showing the usual run-down. The current-voltage relationship of Ba(2+) currents was consistent with that of the high-voltage-activated Ca(2+) channel. The proportion of Ba(2+) currents inhibited by ω-conotoxin GVIA was larger than 80%, indicating that the major subtype is N type. Amplitudes of tail currents of Ca(2+) currents evoked by repetitive pulses at 50 Hz are stable for 1 s. If the major subtype of VGCCs at synaptic terminals is also N type, this property may contribute to the establishment of stable synaptic connections between accessory lobe neurons, which are reported to fire at frequencies higher than 15 Hz, and postsynaptic neurons in the spinal cord.

Nerve injury induces a Gem-GTPase-dependent downregulation of P/Q-type Ca2+ channels contributing to neurite plasticity in dorsal root ganglion neurons.

Small RGK GTPases, Rad, Gem, Rem1, and Rem2, are potent inhibitors of high-voltage-activated (HVA) Ca(2+) channels expressed in heterologous expression systems. However, the role of this regulation has never been clearly demonstrated in the nervous system. Using transcriptional analysis, we show that peripheral nerve injury specifically upregulates Gem in mice dorsal root ganglia. Following nerve injury, protein expression was increased in ganglia and peripheral nerve, mostly under its phosphorylated form. This was confirmed in situ and in vitro in dorsal root ganglia sensory neurons. Knockdown of endogenous Gem, using specific small-interfering RNA (siRNA), increased the HVA Ca(2+) current only in the large-somatic-sized neurons. Combining pharmacological analysis of the HVA Ca(2+) currents together with Gem siRNA-transfection of larger sensory neurons, we demonstrate that only the P/Q-type Ca(2+) channels were enhanced. In vitro analysis of Gem affinity to various CaVβx-CaV2.x complexes and immunocytochemical studies of Gem and CaVβ expression in sensory neurons suggest that the specific inhibition of the P/Q channels relies on both the regionalized upregulation of Gem and the higher sensitivity of the endogenous CaV2.1-CaVβ4 pair in a subset of sensory neurons including the proprioceptors. Finally, pharmacological inhibition of P/Q-type Ca(2+) current reduces neurite branching of regenerating axotomized neurons. Taken together, the present results indicate that a Gem-dependent P/Q-type Ca(2+) current inhibition may contribute to general homeostatic mechanisms following a peripheral nerve injury.

High-sucrose diets in pregnancy alter angiotensin II-mediated pressor response and microvessel tone via the PKC/Cav1.2 pathway in rat offspring.

This study determines the influence of a prenatal high-sucrose (HS) diet on angiotensin II (Ang II)-mediated pressor response and determine the underlying mechanism. Pregnant rats were provided with a 20% sucrose solution diet throughout gestation. Blood pressure and vascular response to Ang II were measured in 5-month-old adult offspring. Currents of L-type Ca(2+) channels (Cav1.2) were measured in smooth muscle cells of small mesenteric arteries from the offspring. Ang II-mediated pressor response was higher in the offspring exposed to prenatal high sugar compared with the control. In mesenteric arteries from the HS offspring, AT1 receptors (AT1R), not AT2 receptors, mediated the increased vasoconstriction; protein kinase C (PKC) antagonist GF109203X suppressed the Ang II-increased vasoconstriction; PKC agonist phorbol 12,13-dibutyrate produced a greater contractile response that was reversed by the Cav1.2 antagonist nifedipine. The expression of PKCα was increased, whereas PKCδ was unchanged; KCl-induced vasoconstriction was stronger and was suppressed by nifedipine; nifedipine also reduced the enhanced vasoconstriction by Ang II. In addition, the Cav1.2 of smooth muscle cells in mesenteric arteries from the HS offspring showed larger current density, although its expression was unchanged. The data suggest that a HS diet during pregnancy alters Ang II-mediated pressor response and microvessel tone acting through the PKC/Cav1.2 pathway in the offspring that may in part be because of alterations in AT1Rs, PKCα and Cav1.2.

Towards computational modelling of the human foetal electrocardiogram: normal sinus rhythm and congenital heart block.

We aim to engineer a computational model of propagation during normal sinus rhythm in the foetal human heart, by modifying models for adult cardiac tissue to match foetal electrocardiogram (fECG) characteristics. The model will be partially validated by fECG data, and applied to explore possible mechanisms of arrhythmogenesis in the foetal heart. Foetal electrocardiograms have been recorded during pregnancy, with P- and T-waves, and the QRS complex, identified by averaging and signal processing. Intervals of the fECG are extracted and used to modify currently available human adult cardiomyocyte models. RR intervals inform models of the pacemaking cells by constraining their rate, the QT interval and its rate dependence constrain models of ventricular cells, and the width of the P-wave, the QR and PR intervals constrain propagation times, conduction velocities, and intercellular coupling. These cell models are coupled into a one-dimensional (1D) model of propagation during normal sinus rhythm in the human foetal heart. We constructed a modular, heterogeneous 1D model for propagation in the foetal heart, and predicted the effects of reduction in L-type Ca(++) current. These include bradycardia and atrioventricular conduction blocks. These may account quantitatively for congenital heart block produced by positive IgG antibodies. The fECG can be interpreted mechanistically and quantitatively by using a simple computational model for propagation. After further validation, by clinical recordings of the fECG and the electrophysiological experiments on foetal cardiac cells and tissues, the model may be used to predict the effects of maternally administered pharmaceuticals on the fECG.

L‐typeCa2+channel current characteristics are preserved in rat tail artery myocytes after one‐day storage

To develop a cheap and simple method of storing for 24-h vascular tissue and single myocytes while preserving therein the biophysical and pharmacological characteristics of L-type Ca(2+) channels and contractile activity. Rings or vascular smooth muscle cells obtained from the rat tail main artery were used either freshly (R0h and VSMC0h) or stored for 24 h (R24h and VSMC24h) at 4 °C, to record whole-cell L-type Ca(2+) currents (IC a(L) ) or measure contractile responses. R0h/VSMC0h and R24h/VSMC24h comparably contracted when stimulated with phenylephrine, high KCl or ATP. In both VSMC0h and VSMC24h, IC a(L) was identified and characterized as a stable inward current for at least 35 min; IC a(L) was comparably inhibited by the Ca(2+) antagonists nifedipine, verapamil and diltiazem and increased by the Ca(2+) channel agonist (S)-(-)-Bay K 8644; current density and current-voltage relationships were similar; at more hyperpolarized holding potentials, IC a(L) intensity increased comparably; nifedipine shifted the steady-state inactivation curve towards more negative potentials, while verapamil blocked IC a(L) in a frequency-dependent manner and slowed down the rate of recovery from inactivation in a comparable way. Findings show that smooth muscle contractile activity and the biophysical and pharmacological features of L-type Ca(2+) channels are similar in VSMC24h and VSMC0h. The fact that reproducible results were obtained in vascular myocytes up to 24 h after dissociation may facilitate vascular smooth muscle cell investigation by increasing throughput and reducing the number of animals required.

Decreased L-type calcium current in antral smooth muscle cells of STZ-induced diabetic rats.

Diabetic gastroparesis (delayed gastric emptying) is associated with antral hypomotility. L-type Ca(2+) channels play an important role in generation of action potentials and activation of contractions. This study was designed to investigate if the function of the L-type Ca(2+) channels of antral circular smooth muscle cells (SMCs) is impaired in streptozotocin (STZ)-induced diabetic rats. Eight weeks after the injection of STZ or vehicle, whole-cell patch clamp was used to record Ca(2+) currents, and isometric tension recording was used to measure Ca(2+) influx-induced contractions in circular muscle strips. Solid gastric emptying was measured in diabetic and control rats. Protein expression of Ca(2+) αlC-subunit in antral smooth muscles was compared between diabetic and control rats. (1) Solid gastric emptying, independent of age or bodyweight, was slower in the diabetic rats, even after acute correction of hyperglycemia. (2) Verapamil, a potent calcium channel blocker, dose dependently reduced solid gastric emptying in normal rats. (3) Current density of L-type Ca(2+) channel at 10 mV in antral circular SMCs was significantly decreased in the diabetic rats (-9.8 ± 0.7 pA/pF vs -15.9 ± 1.0 pA/pF in control, p < 0.001). However, protein expression of the Ca(2+) channel in antral muscles did not differ between diabetic and control rats. (4) Contractile responses to 1 and 3 mM [Ca(2+) ] were significantly reduced in the diabetic antral circular muscle strips, indicative of reduced Ca(2+) influx. These data suggested that the decreased L-type Ca(2+) current in antral SMCs may contribute to antral hypomotility in STZ-induced diabetic rats.

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

CXCL12 chemokine and its receptors as major players in the interactions between immune and nervous systems

The chemokine CXCL12/stromal cell-derived factor 1 alpha has first been described in the immune system where it functions include chemotaxis for lymphocytes and macrophages, migration of hematopoietic cells from fetal liver to bone marrow and the formation of large blood vessels. Among other chemokines, CXCL12 has recently attracted much attention in the brain as it has been shown that it can be produced not only by glial cells but also by neurons. In addition, its receptors CXCR4 and CXCR7, which are belonging to the G protein-coupled receptors family, are abundantly expressed in diverse brain area, CXCR4 being a major co-receptor for human immunodeficiency virus 1 entry. This chemokine system has been shown to play important roles in brain plasticity processes occurring during development but also in the physiology of the brain in normal and pathological conditions. For example, in neurons, CXCR4 stimulation has been shown regulate the synaptic release of glutamate and γ-aminobutyric acid (GABA). It can also act post-synaptically by activating a G protein activated inward rectifier K+ (GIRK), a voltage-gated K channel Kv2.1 associated to neuronal survival, and by increasing high voltage activated Ca2+ currents. In addition, it has been recently evidenced that there are several cross-talks between the CXCL12/CXCR4–7 system and other neurotransmitter systems in the brain (such as GABA, glutamate, opioids, and cannabinoids). Overall, this chemokine system could be one of the key players of the neuro-immune interface that participates in shaping the brain in response to changes in the environment.

How does β-adrenergic signalling affect the transitions from ventricular tachycardia to ventricular fibrillation?

Ventricular tachycardia (VT) and fibrillation (VF) are the most lethal cardiac arrhythmias. The degeneration of VT into VF is associated with the breakup of a spiral wave of the action potential in cardiac tissue. β-Adrenergic (βAR) signalling potentiates the L-type Ca current (ICaL) faster than the slow delayed rectifier potassium current (IKs), which transiently prolongs the action potential duration (APD) and promotes early after depolarizations. In this study, we aimed at investigating how βAR signalling affects the transition from VT to VF. We used a physiologically detailed computer model of the rabbit ventricular myocyte in a two-dimensional tissue to determine how spiral waves respond to βAR activation following administration of isoproterenol. A simplified mathematical model was also used to investigate the underlying dynamics. We found that the spatiotemporal behaviour of spiral waves strongly depends on the kinetics of βAR activation. When βAR activation is rapid, a stable spiral wave turns into small fragments and its electrocardiogram reveals the transition from VT to VF. This is due to the transiently steepened APD restitution induced by the faster activation of ICaL vs. IKs upon sudden βAR activation. The spiral wave may also disappear if its transient wavelength is too large to be supported by the tissue size upon sudden strong βAR activation that prolongs APD transiently. When βAR activation is gradual, a stable spiral wave remains such, because of more limited increase in both APD and slope of APD restitution due to more contemporaneous ICaL and IKs activation. Changes in APD restitution during βAR activation revealed a novel transient spiral wave dynamics; this spatiotemporal characteristic strongly depends on the protocol of isoproterenol application.

Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells

Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca(2+) channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca(2+) currents in mouse ventricular cardiomyocytes (VCM, with Ca(2+) as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba(2+) as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca(2+) channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca(2+) channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.

The T-type calcium channel as a new therapeutic target for Parkinson’s disease

Parkinson's disease (PD) is one of the most prevalent movement disorder caused by degeneration of the dopaminergic neurons in substantia nigra pars compacta. Deep brain stimulation (DBS) at the subthalamic nucleus (STN) has been a new and effective treatment of PD. It is interesting how a neurological disorder caused by the deficiency of a specific chemical substance (i.e., dopamine) from one site could be so successfully treated by a pure physical maneuver (i.e., DBS) at another site. STN neurons could discharge in the single-spike or the burst modes. A significant increase in STN burst discharges has been unequivocally observed in dopamine-deprived conditions such as PD, and was recently shown to have a direct causal relation with parkinsonian symptoms. The occurrence of burst discharges in STN requires enough available T-type Ca(2+) currents, which could bring the relatively negative membrane potential to the threshold of firing Na(+) spikes. DBS, by injection of negative currents into the extracellular space, most likely would depolarize the STN neuron and then inactivate the T-type Ca(2+) channel. Burst discharges are thus decreased and parkinsonian locomotor deficits ameliorated. Conversely, injection of positive currents into STN itself could induce parkinsonian locomotor deficits in animals without dopaminergic lesions. Local application of T-type Ca(2+) channel blockers into STN would also dramatically decrease the burst discharges and improve parkinsonian locomotor symptoms. Notably, zonisamide, which could inhibit T-type Ca(2+) currents in STN, has been shown to benefit PD patients in a clinical trial. From the pathophysiological perspectives, PD can be viewed as a prototypical disorder of "brain arrhythmias". Modulation of relevant ion channels by physical or chemical maneuvers may be important therapeutic considerations for PD and other diseases related to deranged neural rhythms.

Bone morphogenetic protein-4 induces upregulation of Cav3.1 Ca2+ channels in HL-1 atrial myocytes

Cardiac T-type Ca(2+) channels are reexpressed in atrial and ventricular myocytes under various pathological conditions such as post-myocardial infarction, hypertrophy, and heart failure, but relatively little is known about the mechanisms. Our previous study found that bone morphogenetic protein-4 (BMP4) was reexpressed in pathological cardiac hypertrophy models and BMP4-mediated cardiomyocyte hypertrophy. We hypothesized that BMP4 could upregulate cardiac T-type Ca(2+) channels in HL-1 atrial myocytes. The T-type Ca(2+) currents were recorded by using the patch-clamp technique, and the expressions of Cav3.1 and Cav3.2 were measured by real-time PCR method in HL-1 cells. BMP4 and Cav3.1 mRNA expressions increased in the left atrium from the pressure overload-induced hypertrophy of mice hearts. BMP4 treatment for 48 h induced increase of Cav3.1 but not Cav3.2 mRNA expression in HL-1 cells, and the increase was inhibited by BMP4 inhibitor noggin. Acute treatment with BMP4 did not affect T-type Ca(2+) currents, but chronic treatment (48 h) significantly increased the amplitude of T-type Ca(2+) currents in HL-1 cells. Chronic treatment with BMP4 induced upregulation of NADPH oxidase-4 (NOX4), increase of reactive oxygen species (ROS) level, and activation of mitogen-activated protein kinase (MAPK)-activated protein kinases c-jun N-terminal kinases (JNK) and p38. BMP4-induced upregulation of Cav3.1 mRNA was inhibited by NADPH oxidase inhibitor apocynin, the radical scavenger tempol, JNK inhibitor SP600125, and p38 inhibitor SB203580. In conclusion, BMP4 induces upregulation of Cav3.1 Ca(2+) channels and T-type Ca(2+) currents in HL-1 atrial myocytes through ROS/MAPK pathways.

Novel TRPM8 Antagonist Attenuates Cold Hypersensitivity after Peripheral Nerve Injury in Rats

Abnormal cold sensitivity is a common feature of a range of neuropathies. In the murine somatosensory system, multiple aspects of cold sensitivity are dependent on TRPM8, both short term and in response to peripheral nerve injury. The specialized nature of cold-sensitive afferents and the restricted expression of TRPM8 render it an attractive target for the treatment of cold hypersensitivity. This current study examines the effect of a novel TRPM8 antagonist (M8-An) in naive and spinal nerve-ligated rats through behavioral and in vivo electrophysiological approaches. In vitro, M8-An inhibited icilin-evoked Ca(2+) currents in HEK293 cells stably expressing human TRPM8 with an IC(50) of 10.9 nM. In vivo, systemic M8-An transiently decreased core body temperature. Deep dorsal horn recordings were made in vivo from neurons innervating the hind paw. M8-An inhibited neuronal responses to innocuous and noxious cooling of the receptive field in spinal nerve-ligated rats but not in naive rats. No effect on neuronal responses to mechanical and heat stimulation was observed. In addition, M8-An also attenuated behavioral responses to cold but not mechanical stimulation after nerve ligation without affecting the uninjured contralateral response. The data presented here support a contribution of TRPM8 to the pathophysiology of cold hypersensitivity in this model and highlight the potential of the pharmacological block of TRPM8 in alleviating the associated symptoms.

Reduced excitability of gp130-deficient nociceptors is associated with increased voltage-gated potassium currents and Kcna4 channel upregulation

Neuropathic pain and pain arising from local inflammation are characterized by increased release of inflammatory mediators like interleukin-6 (IL-6) by immune cells. The levels of IL-6 is increased in various painfull conditions and correlates with the severity of thermal and mechanical hypersensitivity. Deletion of the IL-6 signal transducer glycoprotein 130 (gp130) reduces inflammation associated with hypersensitivity to thermal and mechanical stimuli. In this study, we show that nociceptor-specific deletion of gp130 alters excitability parameters that are linked to changes in the potassium conductance. In SNS-gp130(-/-) sensory neurons, the resting membrane potential was reduced. Moreover the repolarization speed of the action potential and afterhypolarization was augmented, however, voltage-gated Na(+) and Ca(2+) current were not obviously altered. The main difference between gp130-deficient and control neurons was a significant increase in the conductance of both delayed rectifier as well as A-type potassium currents. Taqman RT-PCR analysis revealed significantly higher levels of Kcna4 mRNA, encoding A-type Kv1.4 potassium channel, in neuron cultures from SNS-gp130(-/-) versus control mice, which may account for the electrophysiological data. No difference in other voltage-gated ion channel mRNAs was observed. The present data show for the first time increased A-type K(+) currents and expression of voltage-gated potassium channel Kcna4 (Kv1.4) in SNS-gp130(-/-) nociceptors. This suggests that gp130 acts as a break for the expression of potassium channels and important regulator hub for nociceptor excitability.

Transcription Factor Sp1 Regulates T‐Type Ca2+ Channel CaV3.1 Gene Expression

Voltage-gated T-type Ca(2+) (CaV 3) channels mediate a number of physiological events in developing and mature cells, and are implicated in neurological and cardiovascular diseases. In mammals, there are three distinct T-channel genes (CACNA1G, CACNA1H, and CACNA1I) encoding proteins (CaV 3.1-CaV 3.3) that differ in their localization as well as in molecular, biophysical, and pharmacological properties. The CACNA1G is a large gene that contains 38 exons and is localized in chromosome 17q22. Only basic characteristics of the CACNA1G gene promoter region have been investigated classifying it as a TATA-less sequence containing several potential transcription factor-binding motifs. Here, we cloned and characterized a proximal promoter region and initiated the analysis of transcription factors that control CaV 3.1 channel expression using the murine Cacna1g gene as a model. We isolated a ∼1.5 kb 5'-upstream region of Cacna1g and verified its transcriptional activity in the mouse neuroblastoma N1E-115 cell line. In silico analysis revealed that this region possesses a TATA-less minimal promoter that includes two potential transcription start sites and four binding sites for the transcription factor Sp1. The ability of one of these sites to interact with the transcription factor was confirmed by electrophoretic mobility shift assays. Consistent with this, Sp1 over-expression enhanced promoter activity while siRNA-mediated Sp1 silencing significantly decreased the level of CaV 3.1 protein and reduced the amplitude of whole-cell T-type Ca(2+) currents expressed in the N1E-115 cells. These results provide new insights into the molecular mechanisms that control CaV 3.1 channel expression.

A Study of Early Afterdepolarizations in a Model for Human Ventricular Tissue

Sudden cardiac death is often caused by cardiac arrhythmias. Recently, special attention has been given to a certain arrhythmogenic condition, the long-QT syndrome, which occurs as a result of genetic mutations or drug toxicity. The underlying mechanisms of arrhythmias, caused by the long-QT syndrome, are not fully understood. However, arrhythmias are often connected to special excitations of cardiac cells, called early afterdepolarizations (EADs), which are depolarizations during the repolarizing phase of the action potential. So far, EADs have been studied mainly in isolated cardiac cells. However, the question on how EADs at the single-cell level can result in fibrillation at the tissue level, especially in human cell models, has not been widely studied yet. In this paper, we study wave patterns that result from single-cell EAD dynamics in a mathematical model for human ventricular cardiac tissue. We induce EADs by modeling experimental conditions which have been shown to evoke EADs at a single-cell level: by an increase of L-type Ca currents and a decrease of the delayed rectifier potassium currents. We show that, at the tissue level and depending on these parameters, three types of abnormal wave patterns emerge. We classify them into two types of spiral fibrillation and one type of oscillatory dynamics. Moreover, we find that the emergent wave patterns can be driven by calcium or sodium currents and we find phase waves in the oscillatory excitation regime. From our simulations we predict that arrhythmias caused by EADs can occur during normal wave propagation and do not require tissue heterogeneities. Experimental verification of our results is possible for experiments at the cell-culture level, where EADs can be induced by an increase of the L-type calcium conductance and by the application of I blockers, and the properties of the emergent patterns can be studied by optical mapping of the voltage and calcium.

Hyperactivation of L-type voltage-gated Ca2+ channels in Caenorhabditis elegans striated muscle can result from point mutations in the IS6 or the IIIS4 segment of the α1 subunit.

Several human diseases, including hypokalemic periodic paralysis and Timothy syndrome, are caused by mutations in voltage-gated calcium channels. The effects of these mutations are not always well understood, partially because of difficulties in expressing these channels in heterologous systems. The use of Caenorhabditis elegans could be an alternative approach to determine the effects of mutations on voltage-gated calcium channel function because all the main types of voltage-gated calcium channels are found in C. elegans, a large panel of mutations already exists and efficient genetic tools are available to engineer customized mutations in any gene. In this study, we characterize the effects of two gain-of-function mutations in egl-19, which encodes the L-type calcium channel α1 subunit. One of these mutations, ad695, leads to the replacement of a hydrophobic residue in the IIIS4 segment. The other mutation, n2368, changes a conserved glycine of IS6 segment; this mutation has been identified in patients with Timothy syndrome. We show that both egl-19 (gain-of-function) mutants have defects in locomotion and morphology that are linked to higher muscle tone. Using in situ electrophysiological approaches in striated muscle cells, we provide evidence that this high muscle tone is due to a shift of the voltage dependency towards negative potentials, associated with a decrease of the inactivation rate of the L-type Ca(2+) current. Moreover, we show that the maximal conductance of the Ca(2+) current is decreased in the strongest mutant egl-19(n2368), and that this decrease is correlated with a mislocalization of the channel.