Calcium Channel Currents Research Articles

Molecular regions underlying the activation of low- and high-voltage activating calcium channels

We have studied two aspects of calcium channel activation. First, we investigated the molecular regions that are important in determining differences in activation between low- and high-voltage activated channels. For this, we made chimeras between the low-voltage activating Ca(V)3.1 channel and the high-voltage activating Ca(V)1.2 channel. Chimeras were expressed in oocytes, and calcium channel currents recorded by voltage clamp. For domain I, we found that the molecular region that is important in determining the voltage dependence of activation comprises the pore regions S5-P as well as P-S6, but surprisingly not the voltage sensor S1-S4 region, which might have been expected to play a major part. By contrast, the smaller, but still significant, modulating effects of domain II on activation properties were due to effects involving both S1-S4 and S5-S6 but not the I/II linker. Second, during channel activation we studied movement of the S4 segment in domain I of one of the chimeras, using cysteine-scanning mutagenesis. The reagent parachloromercuribenzensulfonate inhibited currents for mutants V263, A265, L266 and A268, but not for F269 and V271, and voltage dependence of inhibition for residue V263 indicated S4 movement, which occurred before channel opening. The data indicate movement outwards upon depolarisation so as to expose amino acids up to residue 268 in S4.

C31 enhances voltage-gated calcium channel currents in undifferentiated PC12 cells

C31, consisting of 31 amino acid residues, is generated from the carboxyl terminal fragments (CTFs) of amyloid precursor protein (APP). It has been shown that C31 causes apoptosis in neurons and is present in brains of Alzheimer disease (AD) patients. Using whole-cell patch clamp techniques, we investigated effects of C31 on voltage-gated calcium channel (VGCC) currents and the protective effects of β-estradiol on PC12 cells. The results demonstrated that C31 induced a significant increase of the VGCC currents in PC12 cells, which was blocked by β-estradiol. These results suggest that modulation of intracellular calcium levels by VGCC may in part be involved in C31 induced neuronal death associated with AD.

Modulation of Spontaneous Firing in Rat Subthalamic Neurons by 5-HT Receptor Subtypes

The subthalamic nucleus (STN) is considered to be one of the driving forces in the basal ganglia circuit. The STN is innervated by serotonergic afferents from the raphe nucleus and expresses a variety of 5-HT receptor subtypes. We investigated the effects of 5-HT and 5-HT receptor subtype agonists and antagonists on the firing properties of STN neurons in rat brain slices. We used cell-attached, perforated-patch, and whole cell recording techniques to detect changes in firing frequency and pattern and electrical membrane properties. Due to the depolarization of membrane potential caused by reduced potassium conductance, 5-HT (10 microM) increased the firing frequency of STN neurons without changing their firing pattern. Cadmium failed to occlude the effect of 5-HT on firing frequency. 5-HT had no effect on afterhyperpolarization current. These results indicated that the 5-HT action was not mediated by high-voltage-activated calcium channel currents and calcium-dependent potassium currents. 5-HT had no effect on hyperpolarization-activated cation current (I(H)) amplitude and voltage-dependence of I(H) activation, suggesting that I(H) was not involved in 5-HT-induced excitation. The increased firing by 5-HT was mimicked by 5-HT(2/4) receptor agonist alpha-methyl-5-HT and was partially mimicked by 5-HT2 receptor agonist DOI or 5-HT4 receptor agonist cisapride. The 5-HT action was partially reversed by 5-HT4 receptor antagonist SB 23597-190, 5-HT2 receptor antagonist ketanserin, and 5-HT2C receptor antagonist RS 102221. Our data indicate that 5-HT has significant ability to modulate membrane excitability in STN neurons; modulation is accomplished by decreasing potassium conductance by activating 5-HT4 and 5-HT2C receptors.

Contrasting phenotypes of putative proprioceptive and nociceptive trigeminal neurons innervating jaw muscle in rat.

BackgroundDespite the clinical significance of muscle pain, and the extensive investigation of the properties of muscle afferent fibers, there has been little study of the ion channels on sensory neurons that innervate muscle. In this study, we have fluorescently tagged sensory neurons that innervate the masseter muscle, which is unique because cell bodies for its muscle spindles are in a brainstem nucleus (mesencephalic nucleus of the 5th cranial nerve, MeV) while all its other sensory afferents are in the trigeminal ganglion (TG). We examine the hypothesis that certain molecules proposed to be used selectively by nociceptors fail to express on muscle spindles afferents but appear on other afferents from the same muscle.ResultsMeV muscle afferents perfectly fit expectations of cells with a non-nociceptive sensory modality: Opiates failed to inhibit calcium channel currents (ICa) in 90% of MeV neurons, although ICa were inhibited by GABAB receptor activation. All MeV afferents had brief (1 msec) action potentials driven solely by tetrodotoxin (TTX)-sensitive Na channels and no MeV afferent expressed either of three ion channels (TRPV1, P2X3, and ASIC3) thought to be transducers for nociceptive stimuli, although they did express other ATP and acid-sensing channels. Trigeminal masseter afferents were much more diverse. Virtually all of them expressed at least one, and often several, of the three putative nociceptive transducer channels, but the mix varied from cell to cell. Calcium currents in 80% of the neurons were measurably inhibited by μ-opioids, but the extent of inhibition varied greatly. Almost all TG masseter afferents expressed some TTX-insensitive sodium currents, but the amount compared to TTX sensitive sodium current varied, as did the duration of action potentials.ConclusionMost masseter muscle afferents that are not muscle spindle afferents express molecules that are considered characteristic of nociceptors, but these putative muscle nociceptors are molecularly diverse. This heterogeneity may reflect the mixture of metabosensitive afferents which can also signal noxious stimuli and purely nociceptive afferents characteristic of muscle.

Voltage-Gated Calcium Channels as Targets for the Treatment of Chronic Pain

This review focuses on the importance of voltage-gated calcium channels in modulating and controlling the function of peripheral and central neurons involved in nociceptive processing. We describe the different families of voltage-gated calcium channels that are expressed in pain pathway neurons, how the expression levels of calcium channel currents change in chronic pain conditions, and the validation of N-type, T-type, and P-type calcium channels as targets for the treatment of pain. The molecular mechanism of action is reviewed for the most prominent calcium channel-targeted drugs including gabapentin and ziconotide as well as antiepileptics administered off-label for the treatment of pain. We discuss how the major genetic, functional, and pharmacological differences between subtypes of neuronal calcium channels can be leveraged to identify new molecular targets and to discover and develop new therapeutic agents for the treatment of chronic pain syndromes.

Adenosine A1-receptor modulation of glutamate-induced calcium influx in rat retinal ganglion cells.

Although adenosine receptors (A(1)-Rs and A(2)-Rs) have been identified in the mammalian retina, the role of adenosine in this tissue is not fully understood. The purpose of this work was to investigate the action of adenosine on glutamate-induced calcium influx in rat retinal ganglion cells (RGCs) and to determine whether adenosine modulates RGC voltage-gated calcium channels. Purified RGC cultures were generated from neonatal rats with a two-step panning procedure. Isolated RGCs were loaded with the ratiometric calcium-indicator dye fura-2, and the effect of adenosine (and related agonists and antagonists) on intracellular calcium levels ([Ca(2+)](i)) during exposure to glutamate (10 microM with 10 microM glycine) was assessed. The effect of adenosine on calcium channel currents was also studied in isolated RGCs with whole-cell patch-clamp techniques. In addition, the effect of adenosine on [Ca(2+)](i) was investigated in fura dextran-loaded RGCs in an intact adult rat retina preparation. In isolated RGCs, adenosine (10 and 100 microM) significantly reduced the glutamate-induced increase in [Ca(2+)](i) ( approximately 30%). The effect of adenosine was blocked by the A(1)-R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), but not by the A(2)-R antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). Adenosine (10 microM) inhibited calcium channel currents by 43%, and again this effect was blocked by DPCPX, but not DMPX. Adenosine (100 microM) also significantly reduced the elevation of [Ca(2+)](i) in RGCs in the intact retina during exposure to N-methyl-d-aspartate (NMDA; 100 microM). Adenosine can inhibit glutamate-induced calcium influx and voltage-gated calcium currents in rat RGCs through A(1)-R activation. This work supports a role for adenosine as a neuromodulator of mammalian RGCs.

Nefiracetam Attenuates Methamphetamine‐Induced Discriminative Stimulus Effects in Rats

Nefiracetam has been reported to increase intracellular cyclic AMP levels and enhance calcium channel current. Since the cAMP cascade is involved in the development of drug dependence, we investigated whether nefiracetam attenuates the methamphetamine (MAP)-induced discriminative responses in rats. Nefiracetam (50 mg/kg) inhibited MAP-induced discriminative responses. Furthermore, rolipram, raclopride, and L-745870, all of which can enhance cAMP activity, disrupted MAP-paired lever press of rats. Nifedipine and neomycin, which are blockers of voltage-sensitive calcium channels (VSCCs), decreased MAP-induced discriminative responses. However, pretreatment of these VSCC blockers failed to affect the inhibitory effects of nefiracetam on MAP-induced discriminative responses. Our findings suggested that nefiracetam inhibits MAP-induced discriminative responses, which may be at least partly associated with the changes in intracellular cAMP levels.

Protein and gene expression of Ca2+ channel isoforms in murine colon: effect of inflammation.

L-Type voltage-dependent Ca2+ channels (L-VDCC) mediate calcium influx in response to membrane depolarization and regulate intracellular processes such as contraction, secretion, neurotransmission, and gene expression. Colonic inflammation significantly attenuates calcium currents in smooth muscle; however, the basis for this remains unclear. In this study we examined the protein and mRNA expression of two isoforms of Ca(v)1.2, encoded by either exon la or 1b. Both isoforms were detected by Western blots, immunohistochemistry and RT-PCR in smooth muscle cells. Neither the protein nor mRNA expression measured by real-time PCR of either isoforms was affected in colonic myocytes from dextran sulfate sodium-treated mice. In whole-cell voltage-clamp experiments, the amplitude of the calcium currents were decreased by almost 70% by inflammation. The calcium channel currents were attenuated by 50 +/- 3% by the c-src kinase specific inhibitor, PP2, in control cells but only 19 +/- 7% in cells from inflamed mice. These studies suggest that decreased calcium channel currents following colonic inflammation are not due to decreased expression but may result from altered regulation by the non-receptor cellular tyrosine kinase, c-src kinase.

Multiple actions of extracellular ATP and adenosine on calcium currents mediated by various purinoceptors in neurons of nucleus tractus solitarius.

Whole-cell patch-clamp recordings were performed on freshly dissociated nucleus tractus solitarius (NTS) of rat to determine the action of extracellular adenosine 5'-triphosphate (ATP) and adenosine (ADO) on voltage-dependent calcium channel (VDCC) currents (I(Ca)). Application of ATP and ATP-analog inhibited I(Ca). The rank order of potency of inhibition of I(Ca) was 2-methylthioATP (2-MeSATP) > ATP > adenosine 5'-diphosphate (ADP) >> alpha,beta-methylene ATP (alpha,beta-MeATP) = uridine 5'-triphosphate (UTP). Application of ADO receptor agonists also inhibited I(Ca). The rank order of potency of inhibition of I(Ca) was N(6)-cyclohexyladenosine (CHA) > ADO > 2-(4-(2-carboxyethyl)phenylethylamino)adenosine-5'-N-ethylcarboxamideadenosine (CGS-21680) > N(6)-2-(4-aminophenyl)ethyladenosine (APNEA). Application of prepulse attenuated these inhibition. Both intracellular dialysis of guanosin 5'-O-(2-thiodiphosphate) (GDP-beta-S) and anti-G(i) antibody also attenuated these inhibition. L-, N- and P/Q-type VDCCs were inhibited by ATP. In contrast, N- and P/Q-type VDCCs were inhibited by ADO. In addition to inhibition, application of 100 microM ATP facilitated I(Ca). Intracellular dialysis of GDP-beta-S did not attenuate these facilitations. In conclusion, activation of P2Y purinoceptors inhibits L-, N- and P/Q-types VDCCs via G(i)-protein betagamma subunits. Activation of A(1) and/or A(2) receptors inhibit N- and P/Q-types VDCCs via G(i)-protein betagamma subunits. Activation of P2X purinoceptors facilitates Ca(2+) entry in NTS.

Overexpression of β2-Adrenergic Receptors cAMP-dependent Protein Kinase Phosphorylates and Modulates Slow Delayed Rectifier Potassium Channels Expressed in Murine Heart: EVIDENCE FOR RECEPTOR/CHANNEL CO-LOCALIZATION

The cardiac slow delayed rectifier potassium channel (IKs), comprised of (KCNQ1) and beta (KCNE1) subunits, is regulated by sympathetic nervous stimulation, with activation of beta-adrenergic receptors PKA phosphorylating IKs channels. We examined the effects of 2-adrenergic receptors (beta2-AR) on IKs in cardiac ventricular myocytes from transgenic mice expressing fusion proteins of IKs subunits and hbeta2-ARs. KCNQ1 and beta2-ARs were localized to the same subcellular regions, sharing intimate localization within nanometers of each other. In IKs/B2-AR myocytes, IKs density was increased, and activation shifted in the hyperpolarizing direction; IKs was not further modulated by exposure to isoproterenol, and KCNQ1 was found to be PKA-phosphorylated. Conversely, beta2-AR overexpression did not affect L-type calcium channel current (ICaL) under basal conditions with ICaL remaining responsive to cAMP. These data indicate intimate association of KCNQ1 and beta2-ARs and that beta2-AR signaling can modulate the function of IKs channels under conditions of increased beta2-AR expression, even in the absence of exogenous beta-AR agonist.

Roles of Molecular Regions in Determining Differences between Voltage Dependence of Activation of CaV3.1 and CaV1.2 Calcium Channels

Voltage-dependent calcium channels are classified into low voltage-activated and high voltage-activated channels. We have investigated the molecular basis for this difference in voltage dependence of activation by constructing chimeras between a low voltage-activated channel (Ca(V)3.1) and a high voltage-activated channel (Ca(V)1.2), focusing on steady-state activation properties. Wild type and chimeras were expressed in oocytes, and two-electrode voltage clamp recordings were made of calcium channel currents. Replacement of domains I, III, or IV of the Ca 3.1 channel with the corresponding domain of Ca(V)1.2 led (V)to high voltage-activated channels; for these constructs the current/voltage (I/V) curves were similar to those for Ca(V)1.2 wild type. However, replacement of domain II gave only a small shift to the right of the I/V curve and modulation of the activation kinetics but did not lead to a high voltage-activating channel with an I/V curve like Ca 1.2. We also investigated the role of the voltage sensor (V)S4 by replacing the S4 segment of Ca(V)3.1 with that of Ca 1.2. For domain I, there was no shift in the I/V curve (V)as compared with Ca(V)3.1, and there were relatively small shifts to the right for domains III and IV. Taken together, these results suggest that domains I, III, and IV (rather than domain II) are apparently critical for channel opening and, therefore, contribute strongly to the difference in voltage dependence of activation between Ca 3.1 and Ca(V)1.2. However, the S4 segments in domains I, (V)III, and IV did not account for this difference in voltage dependence.

Overexpressed Ca(v)beta3 inhibits N-type (Cav2.2) calcium channel currents through a hyperpolarizing shift of ultra-slow and closed-state inactivation.

It has been shown that β auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of β3 subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed β3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at “physiological” holding potentials (HPs) of −60 and −80 mV. At a HP of −80 mV, coinjection of various concentrations (0–12.5 ng) of the β3 with Cav2.2α1 and α2δ enhanced the maximum conductance of expressed channels at lower β3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The β3-induced current suppression was reversed at a HP of −120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba2+ (40 mM) as a charge carrier also largely diminished the effect of β3 at −80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and β3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel β3 effect. Steady-state inactivation curves revealed that N-type channels exhibited “closed-state” inactivation without β3, and that β3 caused an ∼40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately −85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of β3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at −80 mV. Similar “ultra-slow” inactivation was observed for N-type channels without β3. Thus, β3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting “ultra-slow” and “closed-state” inactivation properties.

Multiple signal pathways coupling VIP and PACAP receptors to calcium channels in hamster submandibular ganglion neurons

The Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two novel neuropeptides which produce particular biological effects caused by interaction with G-protein-coupled receptors. We have shown in a previous study where VIP and PACAP 38 inhibit voltage-dependent calcium channel (VDCC) currents (I Ca) via G-proteins in hamster submandibular ganglion (SMG) neurons. In this study, we attempt to further characterize the signal transduction pathways of VIP-and PACAP 38-induced modulation of I Ca. Application of 1 μM VIP and PACAP 38 inhibited I Ca by 33.0±3.1% and 36.8±2.6%, respectively (mean±S.E.M., n=8). Application of strong voltage prepulse attenuated PACAP 38-induced inhibition of I Ca. Pretreatment of cAMP dependent protein kinase (PKA) activator attenuated VIP-induced inhibition, but not the PACAP 38-induced inhibition. Intracellular dialysis of the PKA inhibitor attenuated the VIP-induced inhibition, but not the PACAP 38-induced inhibition. Pretreatment of protein kinase C (PKC) activator and inhibitor attenuated VIP-induced inhibition, but not the PACAP 38-induced inhibition. Pretreatment of cholera toxin (CTX) attenuated PACAP 38-induced inhibition of I Ca. These findings indicate that there are multiple signaling pathways in VIP and PACAP 38-induced inhibitions of I Ca: one pathway would be the VPAC 1/VPAC 2 receptors-induced inhibition involving both the PKA and PKC, and another one concerns the PAC 1 receptor-induced inhibition via G s-protein βγ subunits. The VIP-and PACAP 38-induced facilitation of I Ca can be observed in the SMG neurons in addition to inhibiting of I Ca.

Ceramide inhibits L-type calcium channel currents in GH 3 cells

In this study, we investigated the effect of ceramide on the L-type Ca 2+ channel (L-channel) in GH 3 cells. We found that C6-ceramide, but not C6-dihydroceramide, the inactive analogue, had an inhibitory effect on BayK 8644-stimulated GH release. Using patch clamp analysis, C6- and C2-ceramide, but not C6-dihydroceramide, were found to inhibit the L-channel current. Increasing intracellular ceramide level with sphingomyelinase also inhibited the L-channel current. The inhibitory effect of ceramide on the L-channel current was attenuated by calphostin C, a myristolated pseudosubstrate protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor. Combined treatment with lavendustin A and the myristolated PKC inhibitor blocked the effect of ceramide on the L-channel current. These results indicate that ceramide, a lipid messenger of the sphingomyelin pathway, is an important regulator of the L-channel in GH 3 cells and both tyrosine kinase and PKC are involved in this effect of ceramide.

Corrigenda

CorrigendaCorrigendaPublished Online:01 Jan 2004https://doi.org/10.1152/jn.00933.2003Original articlesSodium Currents in Neurons From the Rostroventrolateral Medulla of the RatTaste Response Variability and Temporal Coding in the Nucleus of the Solitary Tract of the RatGABA-Mediated Trophic Effect on Oligodendrocytes Requires Na-K-2Cl Cotransport ActivityVoltage-Gated Calcium Channel Currents in Type I and Type II Hair Cells Isolated From the Rat CristaControl Strategies Correcting Inaccurately Programmed Fingertip Forces: Model Predictions Derived From Human BehaviorMoreSectionsPDF (6 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations Volume 89, June 2003 Pages 2904-2916: Fagergren A, Ekeberg Ö, and Forssberg H. “Control Strategies Correcting Inaccurately Programmed Fingertip Forces: Model Predictions Derived From Human Behavior” ( http://jn.physiology.org/cgi/content/full/89/6/2904). The Digital Object Identifier for this article is 10.1152/jn.00939.2002. This was misrepresented in the print version of this article. However, the electronic version of this article has been corrected and so deviates from the print with regard to this correction. Volume 90, July 2003 Pages 155-164: Bao H, Wong WH, Goldberg JM, and Eatock RA. “Voltage-Gated Calcium Channel Currents in type I and Type II Hair Cells Isolated From the Rat Crista” ( http://jn.physiology.org/cgi/content/full/90/1/155). The Digital Object Identifier for this article is 10.1152/jn.00244.2003. This was misrepresented in the print version of this article. However, the electronic version of this article has been corrected and so deviates from the print with regard to this correction. Volume 90, August 2003 Pages 1257-1265: Wang H, Yan Y, Kintner DB, Lytle C, and Sun D. “GABA-Mediated Trophic Effect on Oligodendrocytes Requires Na-K-2Cl Cotransport Activity” ( http://jn.physiology.org/cgi/content/full/90/2/1257). The Digital Object Identifier for this article is 10.1152/jn.01174.2002. This was misrepresented in the print version of this article. However, the electronic version of this article has been corrected and so deviates from the print with regard to this correction. Volume 90, September 2003 Pages 1418-1431: Di Lorenzo PM and Victor JD. “Taste Response Variability and Temporal Coding in the Nucleus of the Solitary Tract of the Rat” ( http://jn.physiology.org/cgi/content/full/90/3/1418). The Digital Object Identifier for this article is 10.1152/jn.00177.2003. This was misrepresented in the print version of this article. However, the electronic version of this article has been corrected and so deviates from the print with regard to this correction.Pages 1635-1642: Rybak IA, Ptak K, Shevtsova NA, and McCrimmon DR. “Sodium Currents in Neurons From the Rostroventrolateral Medulla of the Rat” ( http://jn.physiology.org/cgi/content/full/90/3/1635). The Digital Object Identifier for this article is 10.1152/jn.00150.2003. This was misrepresented in the print version of this article. However, the electronic version of this article has been corrected and so deviates from the print with regard to this correction.This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformation Related ArticlesSodium Currents in Neurons From the Rostroventrolateral Medulla of the Rat 01 Sep 2003Journal of NeurophysiologyTaste Response Variability and Temporal Coding in the Nucleus of the Solitary Tract of the Rat 01 Sep 2003Journal of NeurophysiologyGABA-Mediated Trophic Effect on Oligodendrocytes Requires Na-K-2Cl Cotransport Activity 01 Aug 2003Journal of NeurophysiologyVoltage-Gated Calcium Channel Currents in Type I and Type II Hair Cells Isolated From the Rat Crista 01 Jul 2003Journal of NeurophysiologyControl Strategies Correcting Inaccurately Programmed Fingertip Forces: Model Predictions Derived From Human Behavior 01 Jun 2003Journal of Neurophysiology More from this issue > Volume 91Issue 1January 2004Pages 589-589 Copyright & PermissionsCopyright © 2004 by the American Physiological Societyhttps://doi.org/10.1152/jn.00933.2003History Published online 1 January 2004 Published in print 1 January 2004 Metrics

Extracellular ATP both inhibits and facilitates calcium channel currents in acutely dissociated rat nucleus tractus solitarius.

The postsynaptic actions of exogenously applied adenosine 5'-triphosphate (ATP) were investigated in nucleus tractus solitarius (NTS) of the rat. Whole cell patch-clamp recordings were used to examine the regulation of voltage-dependent Ca2+ channels (VDCCs) currents (I(Ca)) by ATP in freshly dissociated NTS. Application of ATP inhibited I(Ca) from -905 pA to -741 pA. In addition to this inhibition, application of ATP facilitated I(Ca) from -941 pA to -1,094 pA in other neurons. The data presented here demonstrate for the first time that ATP has both inhibitory and facilitative effects on I(Ca) in NTS. It can be considered that ATP acts as a neurotransmitter in the NTS by having multiple regulatory effects on VDCCs.

Modulation and Pharmacology of Low Voltage-Activated (“T-Type”) Calcium Channels

Although T-type calcium channel currents were observed almost 30 years ago, the genes that encode the pore-forming subunits have only been recently reported. When expressed in heterologous systems, three distinct alpha1 subunits (alpha1G (Cav3.1), alpha1H (Car3.2), and alpha1I (Cav3.3)) conduct T-type currents with insert similar but not identical electrophysiological characteristics that. Alpha 1G, alpha 1H, and alpha 1I transcripts are found throughout neural and nonneural tissues, suggesting multiple types of T-type channels (also called low voltage-activated calcium channels (LVAs)) are coexpressed by many tissues. The study of endogenous LVAs has been hampered by a lack of highly selective antagonists that differentiate between LVA subtypes. Furthermore, many pharmacological agents attenuate currents conducted by LVA and high voltage-activated calcium channels (HVAs). At least 15 classes of pharmacological agents affect T-type currents, and the therapeutic use of many of these drugs has implicated LVAs in the etiology of a variety of diseases. Comparison of the responses of recombinant and native LVAs to pharmacological agents and endogenous modulatory molecules will lead to a better understanding of LVAs in normal and diseased cells.

Modified Cardiovascular L-type Channels in Mice Lacking the Voltage-dependent Ca2+ Channel β3 Subunit

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.

Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart.

The small G protein Ras-mediated signaling pathway has been implicated in the development of hypertrophy and diastolic dysfunction in the heart. Earlier cellular studies have suggested that the Ras pathway is responsible for reduced L-type calcium channel current and sarcoplasmic reticulum (SR) calcium uptake associated with sarcomere disorganization in neonatal cardiomyocytes. In the present study, we investigated the in vivo effects of Ras activation on cellular calcium handling and sarcomere organization in adult ventricular myocytes using a newly established transgenic mouse model with targeted expression of the H-Ras-v12 mutant. The transgenic hearts expressing activated Ras developed significant hypertrophy and postnatal lethal heart failure. In adult ventricular myocytes isolated from the transgenic hearts, the calcium transient was significantly depressed but membrane L-type calcium current was unchanged compared with control littermates. The expressions of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a and phospholamban (PLB) were significantly reduced at mRNA levels. The amount of SERCA2a protein was also modestly reduced. However, the expression of PLB protein and gross sarcomere organization remained unchanged in the hypertrophic Ras hearts, whereas Ser(16) phosphorylation of PLB was dramatically inhibited in the Ras transgenic hearts compared with controls. Hypophosphorylation of PLB was also associated with a significant induction of protein phosphatase 1 expression. Therefore, our results from this in vivo model system suggest that Ras-induced contractile defects do not involve decreased L-type calcium channel activities or disruption of sarcomere structure. Rather, suppressed SR calcium uptake due to reduced SERCA2a expression and hypophosphorylation of PLB due to changes in protein phosphatase expression may play important roles in the diastolic dysfunction of Ras-mediated hypertrophic cardiomyopathy.

Bursting Dynamics of a Model Neuron Induced by Intrinsic Channel Noise

Irregular switching of ion channels is an inherent source of noise in real neurons. Taking into account the stochastic nature of calcium and fast potassium channel currents in the Plant model of a bursting neuron, we found noise-induced coherent bursting even in the case when the deterministic neuron is silent. The bursts keep their regularity in a wide range of membrane sizes. The measure of this coherence, the coefficient of variation of interspike intervals, passes through a minimum at the membrane area near 150 μ m 2. The degree of coherence obtained from power spectra of spike trains increases monotonously with decrease of membrane size.