Subunit Of L-type Calcium Channel Research Articles

Unilateral vestibular deafferentation-induced changes in calcium signaling-related molecules in the rat vestibular nuclear complex

Inquiries into the neurochemical mechanisms of vestibular compensation, a model of lesion-induced neuronal plasticity, reveal the involvement of both voltage-gated Ca 2+ channels (VGCC) and intracellular Ca 2+ signaling. Indeed, our previous microarray analysis showed an up-regulation of some calcium signaling-related genes such as the α2 subunit of L-type calcium channels, calcineurin, and plasma membrane Ca 2+ ATPase 1 (PMCA1) in the ipsilateral vestibular nuclear complex (VNC) following unilateral vestibular deafferentation (UVD). To further elucidate the role of calcium signaling-related molecules in vestibular compensation, we used a quantitative real-time polymerase chain reaction (PCR) method to confirm the microarray results and investigated changes in expression of these molecules at various stages of compensation (6 h to 2 weeks after UVD). We also investigated the changes in gene expression during Bechterew's phenomenon and the effects of a calcineurin inhibitor on vestibular compensation. Real-time PCR showed that genes for the α2 subunit of VGCC, PMCA2, and calcineurin were transiently up-regulated 6 h after UVD in ipsilateral VNC. A subsequent UVD, which induced Bechterew's phenomenon, reproduced a complete mirror image of the changes in gene expressions of PMCA2 and calcineurin seen in the initial UVD, while the α2 subunit of VGCC gene had a trend to increase in VNC ipsilateral to the second lesion. Pre-treatment by FK506, a calcineurin inhibitor, decelerated the vestibular compensation in a dose-dependent manner. Although it is still uncertain whether these changes in gene expression are causally related to the molecular mechanisms of vestibular compensation, this observation suggests that after increasing the Ca 2+ influx into the ipsilateral VNC neurons via up-regulated VGCC, calcineurin may be involved in their synaptic plasticity. Conversely, an up-regulation of PMCA2, a brain-specific Ca 2+ pump, would increase an efflux of Ca 2+ from those neurons and perhaps prevent cell damage following UVD.

Impact of testosterone on cardiac L-type calcium channels and Ca2+ sparks: Acute actions antagonize chronic effects

While androgens generally have been associated with an increased cardiovascular risk, recent studies indicate potential beneficial acute effects of testosterone. However, detailed evaluation of chronic and acute actions of testosterone on the function of cardiac ICa,L and intracellular Ca2+ handling is limited. To clarify this situation we performed whole-cell and single-channel analysis of ICa,L, recordings of Ca2+ sparks, measurements of contractility and quantitative real-time RT-PCR in rat cardiomyocytes following testosterone pretreatment and acute testosterone application. Pretreatment with testosterone 100nM for 24–30h increased whole-cell ICa,L from 3.8±0.8pA/pF (n=10) to 10.1±0.31pA/pF (n=9) at +10mV (p<0.001). Increase of ICa,L density was caused by both, increased expression levels of the alpha 1C subunit of L-type calcium channel and a pronounced increment of the single-channel activity (availability 81.8±3.15% versus 37.1±7.01%; open probability 12.8±3.09% versus 1.0±0.62%, p<0.01). Moreover, testosterone pretreatment significantly increased the frequency of Ca2+ sparks and improved myocytes contractility without altering SR Ca2+ load. All chronic effects could be inhibited by flutamide. In contrast acute testosterone administration significantly reduced ICa,L density. Indeed, on the single-channel level acute testosterone application completely reversed the chronic testosterone-mediated effects, and antagonized the chronic testosterone effects on Ca2+ spark frequency, which was unaffected by flutamide. Thus, testosterone pretreatment activates ICa,L via nuclear receptor-mediated pathways, while testosterone acutely blocks ICa,L in a direct manner. Thus, testosterone chronically affects the basal level of intracellular Ca2+ handling, which in addition rapidly may be modulated by acute changes of hormone levels.

Up-regulated l-type high voltage-gated calcium channels cause increase in diazepam binding inhibitor induced by sustained morphine exposure in mouse cerebrocortical neurons

Mechanisms of increase in diazepam binding inhibitor (DBI) mRNA expression in mouse cerebrocortical neurons after sustained morphine exposure were investigated. Increases in DBI and its mRNA expressions induced by sustained morphine (0.3 μM) exposure for 3 days were completely abolished by naloxone and nifedipine, but not by ω-agatoxin VIA and ω-conotoxin GIVA. Increase in [ 3H]diltiazem binding to the particulate fractions from the morphine-treated neurons was due to increased B max value with no changes in K d value. Western blot analysis on l-type high voltage-gated calcium channel (HVCC) subunits revealed the increased expressions of α1C, α1D, and α2/δ1 subunits and decreased of β4 subunit expression, whereas expression of N- and P/Q-type HVCC subunits was not changed. These results indicate that morphine-induced increase in DBI mRNA expression is mediated via increased Ca 2+ entry through up-regulated l-type HVCCs.

Central core disease is due to RYR1 mutations in more than 90% of patients

Ryanodine receptor 1 (RYR1) gene mutations are associated with central core disease (CCD), multiminicore disease (MmD) and malignant hyperthermia (MH), and have been reported to be responsible for 47-67% of patients with CCD and rare cases with MmD. However, to date, the true frequency and distribution of the mutations along the RYR1 gene have not been determined yet, since mutation screening has been limited to three 'hot spots', with particular attention to the C-terminal region. In this study, 27 unrelated Japanese CCD patients were included. Clinical histories and muscle biopsies were carefully reviewed. We sequenced all the 106 exons encoding RYR1 with their flanking exon-intron boundaries, and identified 20 novel and 3 previously reported heterozygous missense mutations in 25 of the 27 CCD patients (93%), which is a much higher mutation detection rate than that perceived previously. Among them, six were located outside the known 'hot spots'. Sixteen of 27 (59%) CCD patients had mutations in the C-terminal 'hot spot'. Three CCD patients had a probable autosomal recessive disease with two heterozygous mutations. Patients with C-terminal mutations had earlier onset and rather consistent muscle pathology characterized by the presence of distinct cores in almost all type 1 fibres, interstitial fibrosis and type 2 fibre deficiency. In contrast, patients with mutations outside the C-terminal region had milder clinical phenotype and harbour more atypical cores in their muscle fibres. We also sequenced two genes encoding RYR1-associated proteins as candidate causative genes for CCD: the 12 kD FK506-binding protein (FKBP12) and the alpha1 subunit of L-type voltage-dependent calcium channel or dihydropyridine receptor (CACNA1S). However, no mutation was found, suggesting that these genes may not, or only rarely, be responsible for CCD. Our results indicate that CCD may be caused by RYR1 mutations in the majority of patients.

Effect of L-type calcium channel antagonists on spermine-induced CNS excitation in vivo

The ability of nitrendipine, nisoldipine, verapamil and gabapentin to inhibit the development of CNS excitation induced by spermine was assessed in mice. Injection of an excitotoxic dose of spermine (100 μg, i.c.v.) in mice results in worsening tremor that culminates in the development of a fatal tonic convulsion within 8 h of spermine administration. The dihydropyridines, nitrendipine and nisoldipine, which are L-type calcium channel antagonists acting at the α1 subunit, inhibited the development of spermine-induced effects. Verapamil, which also acts at the α1 subunit of the L-type calcium channel, also inhibited the development of spermine-induced CNS excitation. Gabapentin, a postulated L-type calcium channel antagonist interacting at the α2δ subunit, did not inhibit the development of spermine-induced effects. These results show that antagonists of the α1 subunit of L-type calcium channels can effectively inhibit the effects of spermine in vivo. This may highlight the importance of L-type calcium channels in spermine action.

Sites on Calmodulin That Interact with the C-terminal Tail of Cav1.2 Channel

Two fragments of the C-terminal tail of the alpha(1) subunit (CT1, amino acids 1538-1692 and CT2, amino acids 1596-1692) of human cardiac L-type calcium channel (Ca(V)1.2) have been expressed, refolded, and purified. A single Ca(2+)-calmodulin binds to each fragment, and this interaction with Ca(2+)-calmodulin is required for proper folding of the fragment. Ca(2+)-calmodulin, bound to these fragments, is in a more extended conformation than calmodulin bound to a synthetic peptide representing the IQ motif, suggesting that either the conformation of the IQ sequence is different in the context of the longer fragment, or other sequences within CT2 contribute to the binding of calmodulin. NMR amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538-1595 do not contribute to or alter calmodulin binding to amino acids 1596-1692 of Ca(V)1.2. The interaction with CT2 produces the greatest changes in the backbone amides of hydrophobic residues in the N-lobe and hydrophilic residues in the C-lobe of calmodulin and has a greater effect on residues located in Ca(2+) binding loops I and II in the N-lobe relative to loops III and IV in the C-lobe. In conclusion, Ca(2+)-calmodulin assumes a novel conformation when part of a complex with the C-terminal tail of the Ca(V)1.2 alpha(1) subunit that is not duplicated by synthetic peptides corresponding to the putative binding motifs.

The effect of inflammation on ligand binding and density of L-type calcium and erg K-channels in rat myocardium

The purpose of this study was to investigate the effect of inflammation on ligand binding and density of L-type calcium channels (LCC) and erg potassium channels (EKC) in the rat heart. Acute and chronic inflammation were induced, hearts were removed and either their myocytes were isolated or they were homogenized to provide cardiac cell membranes. Equilibrium ligand binding of 3H-nitrendipine (NIT) to LCC and 3H-dofetilide (DOF) to EKC was performed. Direct effect of TNF on ligand binding was examined using normal rat cardiac membranes. Western blotting of LCC was performed to estimate the density of the target protein. The results indicate that inflammation had no significant effects on dissociation constants (KD). However, the maximum binding (Bmax) for NIT was significantly reduced in chronic (38.7 ± 6.8%) and acute (26.1 ± 8.1%) inflammation. Binding of DOF to EKC in isolated myocytes was not affected by inflammation. The presence of TNF did not affect the binding parameters of NIT to LCC. Western blotting revealed that inflammation had no effect on the density of α1C subunit of LCC. In summary, decrease in efficacy of cardiovascular drug seen in inflamed states could be mainly due to reduced binding to their ligands. Reduced binding may explain, in part, the reduced pharmacological response to calcium channel blockers such as verapamil, in spite of higher plasma concentrations, in the presence of inflammatory diseases such as rheumatoid arthritis. Supported by CIHR. Clinical Pharmacology & Therapeutics (2004) 75, P48–P48; doi: 10.1016/j.clpt.2003.11.179

The fragile x mental retardation protein binds and regulates a novel class of mRNAs containing u rich target sequences

Fragile X syndrome is a common form of inherited mental retardation caused by the absence of the fragile X mental retardation protein (FMRP). It has been hypothesized that FMRP is involved in the processing and/or translation of mRNAs. Human and mouse target-mRNAs, containing purine quartets, have previously been identified. By using cDNA-SELEX (systematic evolution of ligands by exponential enrichment), we identified another class of human target-mRNAs which contain U rich sequences. This technique, in contrast to oligonucleotide-based SELEX, allows the identification of FMRP targets directly from mRNA pools. Many of the proteins encoded by the identified FMRP targets have been implicated in neuroplasticity. Steady state levels of target-mRNAs were unchanged in the brain of fragile X mice. However, levels of two target-encoded proteins, an L-type calcium channel subunit and MAP1B, were downregulated in specific brain regions suggesting a defect in the expression of target-encoded proteins in fragile X syndrome.

Increased open probability of single cardiac L-type calcium channels in patients with chronic atrial fibrillation. role of phosphatase 2A.

The L-type calcium channel (LCC) plays a crucial role in the electrical remodeling of atrial fibrillation (AF). AF is associated with reduction of L-type calcium current density, due to a transcriptional downregulation of the pore forming alpha(1c)-subunit of LCC. However, it is unclear, whether this current reduction is related to a decrease in channel number or to alterations in channel function. Hence, we performed a single LCC analysis to assess channel gating and function in human AF. We used the cell-attached patch-clamp technique in isolated atrial human cardiomyocytes of 25 patients with sinus rhythm (SR) and 15 patients with chronic AF. Protein expression of the pore-forming alpha(1c)-subunit of LCC was reduced by 40% in AF. Single channel peak average current was 1.7-fold higher in AF than in SR, due to a 3.1-fold higher open probability of LCC. Since phosphatase 2A (PP2A) is known to preferentially reduce LCC open probability via channel dephosphorylation, we assessed whether PP2A expression or activity is reduced in AF. Okadaic acid, an inhibitor of phosphatases, increased channel open probability in SR, but not in AF. However, Western blot analysis of atrial homogenates of the same patient population revealed unchanged expression of PP2A. Human AF is characterized by increased single LCC activity, due to an increase of channel open probability. The blunted effect of PP2A on LCC as shown by single channel analysis may be related to a reduction of cytosolic PP2A activity or impaired local interaction between PP2A and LCC in AF.

Increased tyrosine phosphorylation of α 1C subunits of L-type voltage-gated calcium channels and interactions among Src/Fyn, PSD-95 and α 1C in rat hippocampus after transient brain ischemia

It has been reported that the Src family kinases-mediated tyrosine phosphorylation of α 1C subunits of L-type voltage-gated calcium channels (L-VGCCs) potentiates the channel currents. In this study, we evaluated the alterations in the tyrosine phosphorylation level of α 1C and in the interactions involving Src/Fyn, α 1C and PSD-95 in the hippocampus after transient (15 min) brain ischemia followed by various times of reperfusion using immunoprecipitation and immunoblotting. Transient brain ischemia was induced by the method of four-vessel occlusion in Sprague–Dawley rats. The tyrosine phosphorylation level of α 1C subunits elevated immediately after brain ischemia. The elevation in phosphorylation sustained for at least 6 h and peaked at 15 min of reperfusion. Transient brain ischemia and reperfusion also caused rapid and sustained increases in the interactions of Src and Fyn with α 1C subunits. More interestingly, co-immunoprecipitation analysis showed that 15 min of reperfusion dramatically increased the interaction between PSD-95 and α 1C and promoted the formation of α 1C-PSD-95-Src complexes, for the first time. The protein levels of α 1C, Src, Fyn and PSD-95 showed no differences at all time points. These results suggest a novel mechanism involving the ischemia/reperfusion-induced recruitment of L-VGCCs, Src and Fyn to the PSD-95 signaling complex that facilitates the tyrosine phosphorylation of α 1C subunits by Src family kinases and may contribute to the up-regulation of L-VGCCs activity in postischemic hippocampus.

L-type voltage-gated calcium channels are involved in the in vivo and in vitro expression of fear conditioning.

Fear conditioning, a behavioral model of fear learning and cue-related anxiety, causes enhanced neuronal transmission in the thalamic to lateral amygdala pathway.(1,2) In the expression phase of learned fear, this increased transmission recorded in vitro is revealed in increased amplitudes of excitatory postsynaptic currents (EPSCs) and occlusion of paired-pulse facilitation (PPF) implicating a presynaptic increase in transmitter release. Here we examined the contribution of L-type calcium channels in fear conditioning. We measured the effect of nimodipine (Nim, 1.5-20 mg/kg), an L-type calcium channel antagonist, on fear-potentiated startle in which startle was assessed in animals receiving paired or unpaired tone and foot shock. Nim administered intraperitoneally blocked fear-potentiated startle but not baseline startle in a dose-dependent manner. We also analyzed the effect of Nim (10 micro M) in vitro on synaptic facilitation of EPSCs and PPF in slices from naïve control, unpaired control, and fear-conditioned animals. In neurons from naïve control animals, Nim had no effect on EPSC amplitude or PPF, but in slices from fear-conditioned rats, Nim reduced EPSC amplitude, suggesting the recruitment of L-type calcium channels within the fear-conditioning pathway. Nim increased PPF in slices from fear-conditioned animals, suggesting that L-type calcium channels may contribute to increased probability of release in fear conditioning. In slices from unpaired animals, Nim decreased synaptic transmission but had little effect on PPF, suggesting that stress or contextual fear learning may induce L-type channel activity in fear-conditioned and unpaired control animal groups. We also analyzed protein expression of the alpha(1C) and alpha(1D) L-type calcium channel subunits isolated from the amygdala and found that alpha(1C) protein was significantly increased in fear-conditioned animals. These findings suggest that L-type calcium channels play a role in the amygdala in cued fear conditioning and have important implications in the treatment of anxiety and in emotional learning and plasticity.

Regionally selective alterations in expression of the α 1D subunit (Ca v1.3) of L-type calcium channels in the hippocampus of aged rats

Calcium currents through the L-type voltage-sensitive calcium channel (L-VSCC) are increased in neurons of area CA1 of the hippocampus in aged rats and rabbits. Furthermore, increases in mRNA for the pore forming subunit α 1D (Ca v1.3) have been observed in the hippocampus of aged rats. We have studied the protein expression of the two pore forming subunits, α 1C (Ca v1.2) and α 1D, of L-VSCCs in the hippocampus of young and aged rats. Here we report selective age-related changes in expression of α 1D in the hippocampus. Specifically, we find that α 1D protein is increased in area CA1 of aged rats while it is decreased in area CA3. Our data suggest that the altered calcium currents seen in aged animals may be due, at least in part, to alterations in the expression of the α 1D subunit of the L-type calcium channel. These findings contribute to our understanding of the mechanisms responsible for changes in calcium homeostasis during aging.

Alpha(1C) (Ca(V)1.2) L-type calcium channel mediates mechanosensitive calcium regulation.

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the alpha(1C) L-type calcium channel subunit (Ca(V)1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a beta(2) calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac alpha(1C) splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the alpha(1C) COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming alpha(1C)-subunit.

Tubular and cellular localization of the cardiac L-type calcium channel in rat kidney

The mRNAs of several types of calcium channels have been identified in intact rat kidney, and L-type calcium channels cause changes in intracellular calcium in primary cultures of distal tubule cells. The aim of this study was to evaluate the tubular and cellular distribution of the alpha1C subunit of the L-type calcium channel in intact kidney. RT-PCR and Northern blot analysis were used to assess the regional abundance of the mRNA of this channel. Immunocytochemistry combined with confocal microscopy and surface biotinylation were applied to determine the tubular and cellular localization of the protein. Northern blot and RT-PCR analysis indicated that the mRNA of the alpha1C subunit of the cardiac L-type calcium channel was present in whole rat kidney, kidney tubules and kidney cell lines. Western blot of lysates from whole kidney, kidney tubules or cell lines revealed bands of approximately 190 kD for the alpha1C subunit and approximately 60 kD for the beta3 subunit. Confocal immunohistochemistry indicated that the alpha1C subunit of this channel was co-expressed in cells of the distal tubule that express calbindin-D28K, but not in intercalated cells. The alpha1C subunit was also highly expressed in both outer and inner medullary collecting ducts. Serial confocal microscopic images or surface biotinylation experiments determined that the channel was predominantly on the basolateral membrane but had some distribution on the apical membrane. The distribution and cellular localization of the alpha1C subunit of cardiac L-type calcium channel suggest it is probably involved in intracellular and membrane calcium signaling.

Growth and Function of the Embryonic Heart Depend upon the Cardiac-Specific L-Type Calcium Channel α1 Subunit

The heart must function from the moment of its embryonic assembly, but the molecular underpinnings of the first heart beat are not known, nor whether function determines form at this early stage. Here, we find by positional cloning that the embryonic lethal island beat ( isl) mutation in zebrafish disrupts the α1C L-type calcium channel subunit (C-LTCC). The isl atrium is relatively normal in size, and individual cells contract chaotically, in a pattern resembling atrial fibrillation. The ventricle is completely silent. Unlike another mutation with a silent ventricle, isl fails to acquire the normal number of myocytes. Thus, calcium signaling via C-LTCC can regulate heart growth independently of contraction, and plays distinctive roles in fashioning both form and function of the two developing chambers.

Parathyroid cells express dihydropyridine-sensitive cation currents and L-type calcium channel subunits.

Parathyroid cells express Ca2+ -conducting currents that are activated by raising the extracellular Ca2+ concentration ([Ca2+]o). We investigated the sensitivity of these currents to dihydropyridines, the expression of voltage-dependent Ca(2+) channel (VDCC) subunits, and the effects of dihydropyridines on the intracellular free [Ca2+] ([Ca2+]i) and secretion in these cells. Dihydropyridine channel antagonists dose dependently suppressed Ca2+ -conducting currents, and agonists partially reversed the inhibitory effects of the antagonists in these cells. From a bovine parathyroid cDNA library, we isolated cDNA fragments encoding parts of an alpha(1S)- and a beta(3)-subunit of L-type Ca(2+) channels. The alpha(1S)-subunit cDNA from the parathyroid represents an alternatively spliced variant lacking exon 29 of the corresponding gene. Northern blot analysis and immunocytochemistry confirmed the presence of transcripts and proteins for alpha(1)- and beta(3)-subunits in the parathyroid gland. The addition of dihydropyridines had no significant effects on high [Ca2+]o-induced changes in [Ca2+]i and parathyroid hormone (PTH) release. Thus our studies indicate that parathyroid cells express alternatively spliced L-type Ca2+ channel subunits, which do not modulate acute intracellular Ca2+ responses or changes in PTH release.

Localization of voltage‐sensitive L‐type calcium channels in the chicken retina

L-type calcium channels have been associated with synaptic transmission in the retina, and are a potential site for modulation of the release of neurotransmitters. The present study documents the immunohistochemical localization of neuronal alpha1 subunits of L-type calcium channels in chicken retina, using antibodies to the alpha1c, alpha1d and alpha1f subunits of L-type calcium channels. The alpha1c-like subunits were localized to Müller cells, with predominantly radial processes, and a prominent band of horizontal processes in the outer plexiform layer. The antibody to alpha1d subunits labelled most, if not all, cell bodies. The antibody to a human alpha1f subunit strongly labelled photoreceptor terminals. Fainter immunoreactivity was detected in the inner segments of the photoreceptors, a subset of amacrine cells, two bands of labelling in the inner plexiform layer and many ganglion cells. The differential cellular distributons of these alpha1-subunits suggests subtle functional differences in their roles at different cellular locations.

Determinants for Calmodulin Binding on Voltage-dependent Ca2+ Channels

Calmodulin, bound to the alpha(1) subunit of the cardiac L-type calcium channel, is required for calcium-dependent inactivation of this channel. Several laboratories have suggested that the site of interaction of calmodulin with the channel is an IQ-like motif in the carboxyl-terminal region of the alpha(1) subunit. Mutations in this IQ motif are linked to L-type Ca(2+) current (I(Ca)) facilitation and inactivation. IQ peptides from L, P/Q, N, and R channels all bind Ca(2+)calmodulin but not Ca(2+)-free calmodulin. Another peptide representing a carboxyl-terminal sequence found only in L-type channels (designated the CB domain) binds Ca(2+)calmodulin and enhances Ca(2+)-dependent I(Ca) facilitation in cardiac myocytes, suggesting the CB domain is functionally important. Calmodulin blocks the binding of an antibody specific for the CB sequence to the skeletal muscle L-type Ca(2+) channel, suggesting that this is a calmodulin binding site on the intact protein. The binding of the IQ and CB peptides to calmodulin appears to be competitive, signifying that the two sequences represent either independent or alternative binding sites for calmodulin rather than both sequences contributing to a single binding site.

Dendritic L-type calcium currents in mouse spinal motoneurons: implications for bistability.

The intrinsic properties of mammalian spinal motoneurons provide them with the capability to produce high rates of sustained firing in response to transient inputs (bistability). Even though it has been suggested that a persistent dendritic calcium current is responsible for the depolarizing drive underlying this firing property, such a current has not been demonstrated in these cells. In this study, calcium currents are recorded from functionally mature mouse spinal motoneurons using somatic whole-cell patch-clamp techniques. Under these conditions a component of the current demonstrated kinetics consistent with a current originating at a site spatially segregated from the soma. In response to step commands this component was seen as a late-onset, low amplitude persistent current whilst in response to depolarizing-repolarizing ramp commands a low voltage clockwise current hysteresis was recorded. Simulations using a neuromorphic motoneuron model could reproduce these currents only if a noninactivating calcium conductance was placed in the dendritic compartments. Pharmacological studies demonstrated that both the late-onset and hysteretic currents demonstrated sensitivity to both dihydropyridines and the L-channel activator FPL-64176. Furthermore, the alpha1D subunits of L-type calcium channels were immunohistochemically demonstrated on motoneuronal dendrites. It is concluded that there are dendritically located L-type channels in mammalian motoneurons capable of mediating a persistent depolarizing drive to the soma and which probably mediate the bistable behaviour of these cells.

L-type voltage-dependent calcium channel alpha-1C subunit mRNA is present in ejaculated human spermatozoa.

The current study adds to the growing body of evidence that RNA is present in mature ejaculated human spermatozoa. We report that a sodium dodecyl sulphate (SDS)/citric acid extraction method is superior to guanidinium isothiocyanate in terms of reproducibility of RNA recovery from motile sperm populations from individual ejaculates. Using the SDS/citric acid method, RNA was recovered from both fresh and frozen-thawed motile spermatozoa. Sperm RNA were used as templates in reverse transcription-polymerase chain reaction (RT-PCR), in an attempt to identify partial RNA transcripts of a highly conserved region within the alpha-1C (pore-forming) subunit of L-type voltage-dependent calcium channels from 11 individual donors. Control reactions employed primers derived from the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence. In nine of the 11 specimens, gene-specific PCR products were obtained with both the GAPDH and alpha-1C primer pairs. DNA sequencing analysis confirmed that the respective spliced transcripts were amplified. The two cases in which no amplification was obtained were attributed to reduced RNA yield. These data are consistent with results from in-situ RT-PCR of rat testis sections indicating that the testis-specific calcium channel of that species was expressed uniformly in all stages of the germinal epithelium, including mature spermatozoa.