Neuronal L-type Calcium Channels Research Articles

Increased Calcium Influx through L-Type Calcium Channels in Hippocampal Neurons with Exogenous Expression of Presenilin-1 ΔE9 Mutant.

Mutations in the PSEN1 gene encoding presenilin-1 (PS1) protein are the most common cause of familial Alzheimer's disease. One of these, deletion of exon 9, results in the production of shortened PS1 protein (PS1ΔE9). Neuronal hyperexcitability and hyperactivation of L-type calcium channels were observed in cellular and animal models of familial Alzheimer's disease. However, the effect of PS1ΔE9 on L-type calcium channels has not been studied before. We demonstrate enhanced calcium entry through L-type calcium channels in hippocampal mouse neurons with exogenous expression of PS1ΔE9. Additionally, we show that the same effect of the exogenous PS1ΔE9 can be observed in cells with predominant expression of L-type calcium channels subunit Cav1.2. Further research is required to unravel molecular mechanisms underlying hyperactivation L-type calcium channels caused by PS1ΔE9 expression.

Development of phenotypic assays for identifying novel blockers of L-type calcium channels in neurons

L-type calcium channels (LTCCs) are highly expressed in the heart and brain and are critical for cardiac and neuronal functions. LTCC-blocking drugs have a long and successful record in the clinic for treating cardiovascular disorders. In contrast, establishment of their efficacy for indications of the central nervous system remains challenging given the tendency of existing LTCC drugs being functionally and mechanistically more selective for peripheral tissues. LTCCs in vivo are large macromolecular complexes consisting of a pore-forming subunit and other modulatory proteins, some of which may be neuro-specific and potentially harbor mechanisms for neuronal selectivity. To exploit the possibility of identifying mechanistically novel and/or neuro-selective blockers, we developed two phenotypic assays—a calcium flux-based primary screening assay and a patch clamp secondary assay, using rat primary cortical cultures. We screened a library comprised of 1278 known bioactive agents and successfully identified a majority of the potent LTCC-blocking drugs in the library. Significantly, we identified a previously unrecognized LTCC blocker with a novel mechanism, which was corroborated by patch clamp and binding studies. As such, these phenotypic assays are robust and represent an important step towards identifying mechanistically novel and neuro-selective LTCC blockers.

L-type calcium channel contributions to intrinsic excitability and synaptic activity during basolateral amygdala postnatal development.

The amygdala contributes toward emotional processes such as fear, anxiety, and social cognition. Furthermore, evidence suggests that increased excitability of basolateral amygdala (BLA) principal neurons underlie certain neuropsychiatric disorders. Gain-of-function mutations in neuronal L-type calcium channels (LTCCs) are linked to neurodevelopmental diseases, including autism spectrum disorders (ASDs). While LTCCs are expressed throughout the BLA, direct evidence for increased LTCC activity affecting BLA excitability and potentially contributing to disease pathophysiology is lacking. In this study, we utilized a pharmacological approach to examine the contributions of LTCCs to BLA principal cell excitability and synaptic activity at immature (postnatal day 7, P7) and juvenile (P21) developmental stages. Acute upregulation of LTCC activity in brain slices by application of the agonist (S)-Bay K 8644 resulted in increased intrinsic excitability properties including firing frequency response, plateau potential, and spike-frequency adaptation selectively in P7 neurons. Contrastingly, for P21 neurons, the main effect of (S)-Bay K 8644 was to enhance burst firing. (S)-Bay K 8644 increased spontaneous inhibitory synaptic currents at both P7 and P21 but did not affect evoked synaptic currents at either stage. (S)-Bay K 8644 did not alter P7 spontaneous excitatory synaptic currents, although it increased current amplitude in P21 neurons. Overall, the results provide support for the notion that alteration of LTCC activity at specific periods of early brain development may lead to functional alterations to neuronal network activity and subsequently contribute to underlying mechanisms of amygdala-related neurological disorders.NEW & NOTEWORTHY The role of L-type calcium channels (LTCCs) in regulating neuronal electrophysiological properties during development remains unclear. We show that in basolateral amygdala principal neurons, an increase of LTCC activity alters both neuronal excitability and synaptic activity. The results also provide evidence for the distinct contributions of LTCCs at different stages of neurodevelopment and shed insight into our understanding of LTCC dysfunction in amygdala-related neurological disorders.

CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p<0.05), (ii) elevation in ΔΨm (p<0.05), (iii) increased OCR and ATP formation (p<0.05), (iv) increased intracellular NO levels (p<0.05), (v) increased mitochondrial ROS production (p<0.05), and (vi) increased susceptibility to rotenone (p<0.05). Treatment with isradipine was able to partially rescue these negative effects of CNTF-ACM (p<0.05). CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

Regulation of Postsynaptic Stability by the L-type Calcium Channel CaV1.3 and its Interaction with PDZ Proteins.

Alterations in dendritic spine morphology and postsynaptic structure are a hallmark of neurological disorders. Particularly spine pruning of striatal medium spiny neurons and aberrant rewiring of corticostriatal synapses have been associated with the pathology of Parkinson’s disease and L-DOPA induced dyskinesia, respectively. Owing to its low activation threshold the neuronal L-type calcium channel CaV1.3 is particularly critical in the control of neuronal excitability and thus in the calcium-dependent regulation of neuronal functions. CaV1.3 channels are located in dendritic spines and contain a C-terminal class 1 PDZ domain-binding sequence. Until today the postsynaptic PDZ domain proteins shank, densin-180, and erbin have been shown to interact with CaV1.3 channels and to modulate their current properties. Interestingly experimental evidence suggests an involvement of all three PDZ proteins as well as CaV1.3 itself in regulating dendritic and postsynaptic morphology. Here we briefly review the importance of CaV1.3 and its proposed interactions with PDZ proteins for the stability of dendritic spines. With a special focus on the pathology associated with Parkinson’s disease, we discuss the hypothesis that CaV1.3 L-type calcium channels may be critical modulators of dendritic spine stability.

Convergent Modulations by Carboxyl-Termini across L-Type Calcium Channel Subtypes

L-type calcium channels (LTCCs), also known as CaV1 family, are subject to diverse perturbations while acting as signaling hubs in cells, including autonomous modulations by the distal carboxyl terminus (DCT) of its own. Previous studies show that DCT of CaV1.3 and CaV1.4 (i.e., DCTD and DCTF respectively) competes with calmodulin (CaM) for IQ and IQ vicinity (IQ-IQV) of the channel, which results in attenuation of calcium dependent inactivation (CDI) (Liu et al. 2010). In parallel, DCTC (i.e., DCT of CaV1.2) could autonomously inhibit voltage-gated activation (VGA) of the channel (Hulme et al. 2006). Here, our data demonstrate that distal carboxyl-terminus regulatory domain (DCRD) across CaV1 family could mediate both inhibition of CDI (I-CDI) and inhibition of VGA (I-VGA), suggesting a unified scheme of competitive autoregulation by DCRD across CaV1 family. Thus, the apparent discrepancy in phenotypes of I-CDI and/or I-VGA among CaV1 subtypes can be largely attributed to the different configurations of DCT/IQ-IQV complex. Under such scheme, in addition to well-documented I-VGA, the proteolyzed carboxyl-terminus (CCT) of LTCC in neurons could also induce I-CDI. Both I-VGA and I-CDI play crucial roles in CaV1 signaling in neurons. Our results here provide converged mechanisms across CaV1 channel subtypes in their DCT modulations, highlighting the importance of comparative studies in the context of whole-family of CaV1 channels. Moreover, this study also reveals innovative features from CaV1.1 and CaV1.2, inviting further investigations into mechanism and physiology of DCTs.

AKAP-Anchored PKA Maintains Neuronal L-type Calcium Channel Activity and NFAT Transcriptional Signaling

L-type voltage-gated Ca2+ channels (LTCC) couple neuronal excitation to gene transcription. LTCC activity is elevated by the cyclic AMP (cAMP)-dependent protein kinase (PKA) and depressed by the Ca2+-dependent phosphatase calcineurin (CaN), and both enzymes are localized to the channel by A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T cells (NFAT). We report here that the basal activity of AKAP79/150-anchored PKA maintains neuronal LTCC coupling to CaN-NFAT signaling by preserving LTCC phosphorylation in opposition to anchored CaN. Genetic disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of postsynaptic dendritic spines, profound decreases in LTCC phosphorylation and Ca2+ influx, and impaired NFAT movement to the nucleus and activation of transcription. Thus, LTCC-NFAT transcriptional signaling in neurons requires precise organization and balancing of PKA and CaN activities in the channel nanoenvironment, which is only made possible by AKAP79/150 scaffolding.

Raised Activity of L-Type Calcium Channels Renders Neurons Prone to Form Paroxysmal Depolarization Shifts

Neuronal L-type voltage-gated calcium channels (LTCCs) are involved in several physiological functions, but increased activity of LTCCs has been linked to pathology. Due to the coupling of LTCC-mediated Ca2+ influx to Ca2+-dependent conductances, such as KCa or non-specific cation channels, LTCCs act as important regulators of neuronal excitability. Augmentation of after-hyperpolarizations may be one mechanism that shows how elevated LTCC activity can lead to neurological malfunctions. However, little is known about other impacts on electrical discharge activity. We used pharmacological up-regulation of LTCCs to address this issue on primary rat hippocampal neurons. Potentiation of LTCCs with Bay K8644 enhanced excitatory postsynaptic potentials to various degrees and eventually resulted in paroxysmal depolarization shifts (PDS). Under conditions of disturbed Ca2+ homeostasis, PDS were evoked frequently upon LTCC potentiation. Exposing the neurons to oxidative stress using hydrogen peroxide also induced LTCC-dependent PDS. Hence, raising LTCC activity had unidirectional effects on brief electrical signals and increased the likeliness of epileptiform events. However, long-lasting seizure-like activity induced by various pharmacological means was affected by Bay K8644 in a bimodal manner, with increases in one group of neurons and decreases in another group. In each group, isradipine exerted the opposite effect. This suggests that therapeutic reduction in LTCC activity may have little beneficial or even adverse effects on long-lasting abnormal discharge activities. However, our data identify enhanced activity of LTCCs as one precipitating cause of PDS. Because evidence is continuously accumulating that PDS represent important elements in neuropathogenesis, LTCCs may provide valuable targets for neuroprophylactic therapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-013-8234-1) contains supplementary material, which is available to authorized users.

Reduction in neuronal L-type calcium channel activity in a double knock-in mouse model of Alzheimer's disease

Increased function of neuronal L-type voltage-sensitive Ca2+ channels (L-VSCCs) is strongly linked to impaired memory and altered hippocampal synaptic plasticity in aged rats. However, no studies have directly assessed L-VSCC function in any of the common mouse models of Alzheimer's disease where neurologic deficits are typically more robust. Here, we used cell-attached patch-clamp recording techniques to measure L-VSCC activity in CA1 pyramidal neurons of partially dissociated hippocampal “zipper” slices prepared from 14-month-old wild-type mice and memory-impaired APP/PS1 double knock-in mice. Surprisingly, the functional channel density of L-VSCCs was significantly reduced in the APP/PS1 group. No differences in voltage dependency and unitary conductance of L-VSCCs were observed. The results suggest that mechanisms for Ca2+ dysregulation can differ substantially between animal models of normal aging and models of pathological aging.

Dynamic interplay of excitatory and inhibitory coupling modes of neuronal L-type calcium channels

L-type voltage-gated calcium channels (LTCCs) have long been considered as crucial regulators of neuronal excitability. This role is thought to rely largely on coupling of LTCC-mediated Ca(2+) influx to Ca(2+)-dependent conductances, namely Ca(2+)-dependent K(+) (K(Ca)) channels and nonspecific cation (CAN) channels, which mediate afterhyperpolarizations (AHPs) and afterdepolarizations (ADPs), respectively. However, in which manner LTCCs, K(Ca) channels, and CAN channels co-operate remained scarcely known. In this study, we examined how activation of LTCCs affects neuronal depolarizations and analyzed the contribution of Ca(2+)-dependent potassium- and cation-conductances. With the use of hippocampal neurons in primary culture, pulsed current-injections were applied in the presence of tetrodotoxin (TTX) for stepwise depolarization and the availability of LTCCs was modulated by BAY K 8644 and isradipine. By varying pulse length and current strength, we found that weak depolarizing stimuli tend to be enhanced by LTCC activation, whereas in the course of stronger depolarizations LTCCs counteract excitation. Both effect modes appear to involve the same channels that mediate ADP and AHP, respectively. Indeed, ADPs were activated at lower stimulation levels than AHPs. In the absence of TTX, activation of LTCCs prolonged or shortened burst firing, depending on the initial burst duration, and invariably augmented brief unprovoked (such as excitatory postsynaptic potentials) and provoked electrical events. Hence, regulation of membrane excitability by LTCCs involves synchronous activity of both excitatory and inhibitory Ca(2+)-activated ion channels. The overall enhancing or dampening effect of LTCC stimulation on excitability does not only depend on the relative abundance of the respective coupling partner but also on the stimulus intensity.

The interplay of excitatory and inhibitory coupling modes is crucial for the regulation of neuronal electrical activities by L-type calcium channels

Background Neuronal L-type voltage-gated calcium channels (LTCCs) have long been implicated in the regulation of excitability. This function appears to be related to the coupling of LTCC-mediated Ca influx to Ca-dependent conductances, such as KCa channels, e.g. KV2.x (SK), and nonspecific cation (CAN) channels. However, despite numerous data related to the molecular functioning of LTCCs, little is known about the actual role of these channels in cellular electrical excitation. In this study, we examined how activation of LTCCs affects neuronal depolarizations and analyzed the contribution of Ca-dependent potassium and cation conductances.

Knockdown of L Calcium Channel Subtypes: Differential Effects in Neuropathic Pain

The maintenance of chronic pain states requires the regulation of gene expression, which relies on an influx of calcium. Calcium influx through neuronal L-type voltage-gated calcium channels (LTCs) plays a pivotal role in excitation-transcription coupling, but the involvement of LTCs in chronic pain remains unclear. We used a peptide nucleic acid (transportan 10-PNA conjugates)-based antisense strategy to investigate the role of the LTC subtypes Ca(V)1.2 and Ca(V)1.3 in long-term pain sensitization in a rat model of neuropathy (spinal nerve ligation). Our results demonstrate that specific knockdown of Ca(V)1.2 in the spinal dorsal horn reversed the neuropathy-associated mechanical hypersensitivity and the hyperexcitability and increased responsiveness of dorsal horn neurons. Intrathecal application of anti-Ca(V)1.2 siRNAs confirmed the preceding results. We also demonstrated an upregulation of Ca(V)1.2 mRNA and protein in neuropathic animals concomitant to specific Ca(V)1.2-dependent phosphorylation of the cAMP response element (CRE)-binding protein (CREB) transcription factor. Moreover, spinal nerve ligation animals showed enhanced transcription of the CREB/CRE-dependent gene COX-2 (cyclooxygenase 2), which also depends strictly on Ca(V)1.2 activation. We propose that L-type calcium channels in the spinal dorsal horn play an important role in pain processing, and that the maintenance of chronic neuropathic pain depends specifically on channels comprising Ca(V)1.2.

L-type voltage-gated calcium channels in hippocampal neurons and their potential as anti-epilept(ogen)ic drug targets

Background Neuronal L-type voltage-gated calcium channels (LTCCs) were shown to be involved in the control of neuronal excitability, synaptic plasticity and gene expression. These mechanisms are altered in epileptic tissue and are thought to contribute to epileptogenesis. Hence, LTCCs are interesting targets for epileptic and anti-epileptic therapy. However, their role in epilepsy, whether LTCCs enhance or reduce epileptiform/epileptogenic activity, remained unclear. The aim of this study was to identify in which manner LTCCs contribute and/or modulate electrical excitation.

AKAP79/150 Anchoring of Calcineurin Controls Neuronal L-Type Ca 2+ Channel Activity and Nuclear Signaling

Neuronal L-type calcium channels contribute to dendritic excitability and activity-dependent changes in gene expression that influence synaptic strength. Phosphorylation-mediated enhancement of L-type channels containing the CaV1.2 pore-forming subunit is promoted by A-kinase anchoring proteins (AKAPs) that target cAMP-dependent protein kinase (PKA) to the channel. Although PKA increases L-type channel activity in dendrites and dendritic spines, the mechanism of enhancement in neurons remains poorly understood. Here, we show that CaV1.2 interacts directly with AKAP79/150, which binds both PKA and the Ca2+/calmodulin-activated phosphatase calcineurin (CaN). Cotargeting of PKA and CaN by AKAP79/150 confers bidirectional regulation of L-type current amplitude in transfected HEK293 cells and hippocampal neurons. However, anchored CaN dominantly suppresses PKA enhancement of the channel. Additionally, activation of the transcription factor NFATc4 via local Ca2+ influx through L-type channels requires AKAP79/150, suggesting that this signaling complex promotes neuronal L channel signaling to the nucleus through NFATc4.

Neuronal L-Type Calcium Channels Open Quickly and Are Inhibited Slowly

Neuronal L-type calcium channels are essential for regulating activity-dependent gene expression, but they are thought to open too slowly to contribute to action potential-dependent calcium entry. A complication of studying native L-type channels is that they represent a minor fraction of the whole-cell calcium current in most neurons. Dihydropyridine antagonists are therefore widely used to establish the contribution of L-type channels to various neuronal processes and to study their underlying biophysical properties. The effectiveness of these antagonists on L-type channels, however, varies with stimulus and channel subtype. Here, we study recombinant neuronal L-type calcium channels, CaV1.2 and CaV1.3. We show that these channels open with fast kinetics and carry substantial calcium entry in response to individual action potential waveforms, contrary to most studies of native L-type currents. Neuronal CaV1.3 L-type channels were as efficient as CaV2.2 N-type channels at supporting calcium entry during action potential-like stimuli. We conclude that the apparent slow activation of native L-type currents and their lack of contribution to single action potentials reflect the state-dependent nature of the dihydropyridine antagonists used to study them, not the underlying properties of L-type channels.

Spontaneous muscle action potentials are blocked by N-type and P/Q-calcium channels blockers in the rat spinal cord–muscle co-culture system

This study investigated the effects of the calcium channel blockers nicardipine, calcicludine, ω-conotoxin GVIA, ω-agatoxin IVA, SNX-482, and NiCl on spontaneous muscle action potential of a rat spinal cord–muscle co-culture system. Spontaneous muscle action potential of the innervated muscle cells was blocked by d-tubocurarine (1 μM), but was not significantly affected by the L-type channel blocker nicardipine (100 nM). The neuronal L-type calcium channel blocker, calcicludine (50 and 100 nM), also had no effect on the frequency of spontaneous muscle action potential. However, nicardipine (100 nM) and calcicludine (100 nM) significantly increased the amplitude of muscle action potential. Application of the N-type calcium channel blocker, ω-conotoxin GVIA (50 and 100 nM), and the P/Q-type calcium channel blocker, ω-agatoxin IVA (10, 30, 50, and 100 nM), blocked the frequency and amplitude of spontaneous muscle action potential of the spinal cord–muscle co-cultured cells. In contrast, spontaneous muscle action potential was not affected by the R-type calcium channel blockers SNX-482 (100 nM) or NiCl (500 nM). These results indicate that blockers of N- and P/Q-type voltage-dependent calcium channels inhibit transmitter release at neuromuscular junctions in the rat spinal cord–muscle co-culture system.

L-type calcium channels: the low down.

L-type calcium channels couple membrane depolarization in neurons to numerous processes including gene expression, synaptic efficacy, and cell survival. To establish the contribution of L-type calcium channels to various signaling cascades, investigators have relied on their unique pharmacological sensitivity to dihydropyridines. The traditional view of dihydropyridine-sensitive L-type calcium channels is that they are high-voltage-activating and have slow activation kinetics. These properties limit the involvement of L-type calcium channels to neuronal functions triggered by strong and sustained depolarizations. This review highlights literature, both long-standing and recent, that points to significant functional diversity among L-type calcium channels expressed in neurons and other excitable cells. Past literature contains several reports of low-voltage-activated neuronal L-type calcium channels that parallel the unique properties of recently cloned CaV1.3 L-type channels. The fast kinetics and low activation thresholds of CaV1.3 channels stand in stark contrast to criteria currently used to describe L-type calcium channels. A more accurate view of neuronal L-type calcium channels encompasses a broad range of activation thresholds and recognizes their potential contribution to signaling cascades triggered by subthreshold depolarizations.

Neurological phenotype and synaptic function in mice lacking the Ca V1.3 α subunit of neuronal L-type voltage-dependent Ca 2+ channels

Neuronal L-type calcium channels have been implicated in pain perception and neuronal synaptic plasticity. To investigate this we have examined the effect of disrupting the gene encoding the Ca V1.3 (α1D) α subunit of L-type Ca 2+ channels on neurological function, acute nociceptive behavior, and hippocampal synaptic function in mice. Ca V1.3 α1 subunit knockout (Ca V1.3α1 −/−) mice had relatively normal neurological function with the exception of reduced auditory evoked behavioral responses and lower body weight. Baseline thermal and mechanical thresholds were unaltered in these animals. Ca V1.3α1 −/− mice were also examined for differences in N-methyl- d-aspartate (NMDA) receptor-dependent (100 Hz tetanization for 1 s) and NMDA receptor-independent (200 Hz in 100 μM DL-2-amino-5-phosphopentanoic acid) long-term potentiation within the CA1 region of the hippocampus. Both NMDA receptor-dependent and NMDA receptor-independent forms of long-term potentiation were expressed normally. Radioligand binding studies revealed that the density of (+)[ 3H]isradipine binding sites in brain homogenates was reduced by 20–25% in Ca V1.3α1 −/− mice, without any detectable change in Ca V1.2 (α1C) protein levels as detected using Western blot analysis. Taken together these data indicate that following loss of Ca V1.3α1 subunit expression there is sufficient residual activity of other Ca 2+ channel subtypes to support NMDA receptor-independent long-term potentiation and some forms of sensory behavior/function.

Cav1.4α1 Subunits Can Form Slowly Inactivating Dihydropyridine-Sensitive L-Type Ca2+Channels Lacking Ca2+-Dependent Inactivation

The neuronal L-type calcium channels (LTCCs) Cav1.2alpha1 and Cav1.3alpha1 are functionally distinct. Cav1.3alpha1 activates at lower voltages and inactivates more slowly than Cav1.2alpha1, making it suitable to support sustained L-type Ca2+ inward currents (ICa,L) and serve in pacemaker functions. We compared the biophysical and pharmacological properties of human retinal Cav1.4alpha1 using the whole-cell patch-clamp technique after heterologous expression in tsA-201 cells with other L-type alpha1 subunits. Cav1.4alpha1-mediated inward Ba2+ currents (IBa) required the coexpression of alpha2delta1 and beta3 or beta2a subunits and were detected in a lower proportion of transfected cells than Cav1.3alpha1. IBa activated at more negative voltages (5% activation threshold; -39mV; 15 mm Ba2+) than Cav1.2alpha1 and slightly more positive than Cav1.3alpha1. Voltage-dependent inactivation of IBa was slower than for Cav1.2alpha1 and Cav1.3alpha1( approximately 50% inactivation after 5 sec; alpha2delta1 + beta3 coexpression). Inactivation was not increased with Ca2+ as the charge carrier, indicating the absence of Ca2+-dependent inactivation. Cav1.4alpha1 exhibited voltage-dependent, G-protein-independent facilitation by strong depolarizing pulses. The dihydropyridine (DHP)-antagonist isradipine blocked Cav1.4alpha1 with approximately 15-fold lower sensitivity than Cav1.2alpha1 and in a voltage-dependent manner. Strong stimulation by the DHP BayK 8644 was found despite the substitution of an otherwise L-type channel-specific tyrosine residue in position 1414 (repeat IVS6) by a phenylalanine. Cav1.4alpha1 + alpha2delta1 + beta channel complexes can form LTCCs with intermediate DHP antagonist sensitivity lacking Ca2+-dependent inactivation. Their biophysical properties should enable them to contribute to sustained ICa,L at negative potentials, such as required for tonic neurotransmitter release in sensory cells and plateau potentials in spiking neurons.

Effects of methylmercury on human neuronal L-type calcium channels transiently expressed in human embryonic kidney cells (HEK-293).

Methylmercury (MeHg) disrupts the function of native, high voltage-activated neuronal Ca(2+) channels in several types of cells. However, the effects of MeHg on isolated Ca(2+) channel phenotypes have not been examined. The aim of the present study was to examine the action of MeHg on recombinant, neuronal L-type voltage-sensitive Ca(2+) channels. Human embryonic kidney cells (HEK-293) were transfected with human neuronal cDNA clones of the alpha(1C-1) subunit in combination with alpha(2b) and beta(3a) Ca(2+) channel subunits and the reporter jellyfish green fluorescent protein for transient expression. Current from expressed channels (I(Ba)) and their response to MeHg applied acutely were measured using whole-cell voltage-clamp recording techniques and Ba(2+) (5 mM) as charge carrier. Amplitude of I(Ba) in these cells was reduced by the dihydropyridine (DHP), nimodipine, and enhanced by Bay K8644 [S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3 pyridine carboxylic acid methyl ester]. MeHg (0.125-5.0 microM) caused a time- and concentration-dependent reduction in amplitude of the peak and sustained current through these channels. However, even at the highest concentration of MeHg tested, reduction of current amplitude by MeHg was incomplete. Washing with MeHg-free solution could not reverse its effects. The steady-state inactivation curve was unaltered by MeHg. Increasing the stimulation frequency or the extracellular Ba(2+) concentration each attenuated slightly the reduction in amplitude of I(Ba) by MeHg. In the presence of MeHg (5.0 microM), Bay K8644 still increased the remaining current, and nimodipine (10 microM) reduced residual current that was resistant to MeHg. Thus, although MeHg reduces the amplitude of recombinant, heterologously expressed L-type channel current, a portion of current is resistant to reduction by MeHg. Furthermore, DHP agonists and antagonists retain their ability to affect L-type Ca(2+) channel current even in the presence of MeHg.