Calcium Channel Currents Research Articles

Atrial Fibrillation Activates AMP-Dependent Protein Kinase and its Regulation of Cellular Calcium Handling: Potential Role in Metabolic Adaptation and Prevention of Progression

BackgroundAtrial fibrillation (AF) is associated with metabolic stress, which activates adenosine monophosphate-regulated protein kinase (AMPK). ObjectivesThis study sought to examine AMPK response to AF and associated metabolic stress, along with consequences for atrial cardiomyocyte Ca2+ handling. MethodsCalcium ion (Ca2+) transients (CaTs) and cell shortening (CS) were measured in dog and human atrial cardiomyocytes. AMPK phosphorylation and AMPK association with Ca2+-handling proteins were evaluated by immunoblotting and immunoprecipitation. ResultsCaT amplitude and CS decreased at 4-min glycolysis inhibition (GI) but returned to baseline at 8 min, suggesting cellular adaptation to metabolic stress, potentially due to AMPK activation. GI increased AMPK-activating phosphorylation, and an AMPK inhibitor, compound C (CompC), abolished the adaptation of CaT and CS to GI. The AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) increased CaT amplitude and CS, restoring CompC-induced CaT and CS decreases. CompC decreased L-type calcium channel current (ICa,L), along with ICa,L-triggered CaT amplitude and sarcoplasmic reticulum (SR) Ca2+ content under voltage clamp conditions in dog cells and suppressed CaT and ICa,L in human cardiomyocytes. Small interfering ribonucleic acid-based AMPK knockdown decreased CaT amplitude in neonatal rat cardiomyocytes. L-type Ca2+ channel α subunits coimmunoprecipitated with AMPKα. Atrial AMPK-activating phosphorylation was enhanced by 1 week of electrically maintained AF in dogs; fractional AMPK phosphorylation was increased in paroxysmal AF and reduced in longstanding persistent AF patients. ConclusionsAMPK is activated by metabolic stress and AF, and helps maintain the intactness of atrial ICa,L, Ca2+ handling, and cell contractility. AMPK contributes to the atrial compensatory response to AF-related metabolic stress; AF-related metabolic responses may be an interesting new therapeutic target.

Brugada Syndrome: Clinical, Genetic, Molecular, Cellular, and Ionic Aspects

Brugada syndrome (BrS) is an inherited cardiac arrhythmia syndrome first described as a new clinical entity in 1992. Electrocardiographically characterized by distinct coved type ST segment elevation in the right-precordial leads, the syndrome is associated with a high risk for sudden cardiac death in young adults, and less frequently in infants and children. The electrocardiographic manifestations of BrS are often concealed and may be unmasked or aggravated by sodium channel blockers, a febrile state, vagotonic agents, as well as by tricyclic and tetracyclic antidepressants. An implantable cardioverter defibrillator is the most widely accepted approach to therapy. Pharmacologic therapy is designed to produce an inward shift in the balance of currents active during the early phases of the right ventricular action potential (AP) and can be used to abort electrical storms or as an adjunct or alternative to device therapy when use of an implantable cardioverter defibrillator is not possible. Isoproterenol, cilostazol, and milrinone boost calcium channel current and drugs like quinidine, bepridil, and the Chinese herb extract Wenxin Keli inhibit the transient outward current, acting to diminish the AP notch and thus to suppress the substrate and trigger for ventricular tachycardia or fibrillation. Radiofrequency ablation of the right ventricular outflow tract epicardium of patients with BrS has recently been shown to reduce arrhythmia vulnerability and the electrocardiographic manifestation of the disease, presumably by destroying the cells with more prominent AP notch. This review provides an overview of the clinical, genetic, molecular, and cellular aspects of BrS as well as the approach to therapy.

Role of α-synuclein in cognitive dysfunction: Studies in Drosophila melanogaster.

α-Synuclein (α-Syn) is hypothesized to have a critical role in sporadic and genetic cases of Parkinson's disease (PD) in which Lewy bodies, as the hallmark of PD, are formed from abnormal aggregates of α-Syn. To determine the role of α-Syn in the motor and cognitive dysfunction observed in PD, a Drosophila melanogaster model was established to investigate the electrophysiological and ethological changes caused by overexpression of α-Syn. The present data indicated that α-Syn overexpression reduced the synaptic transmission of cholinergic neurons by modulating the calcium channel currents in the projection neurons in the antennal lobe region of the Drosophila brain, as well as the learning and memory ability of the flies. However, the locomotor ability of the Drosophila remained unaffected. The present findings suggested that α-Syn may be associated with senile dementia in patients with PD.

ZC88, a novel N-type calcium channel blocker from 4-amino-piperidine derivatives state-dependent inhibits Cav2.2 calcium channels

Small molecular inhibitors of Cav2.2 have been reported for the treatment of neuropathic pain; however, low selectivity and side effects limit their further development. In our study, a series of new compounds were designed and synthesized by optimizing the 4-amino-piperidine template. The results show that ZC88 inhibits transiently expressed Cav2.2 in state-dependent manner in oocytes with an IC50 of 0.45±0.09μM. The steady-state inactivation relationship curve is shifted to more negative potentials for the calcium channels, suggesting that ZC88 blocks inactivated state of the channel. ZC88 does not present any remarkable effects on voltage-gated P/Q-type calcium channel currents, l-type calcium channel currents, potassium channel and sodium channel currents. Taken together, these in vitro data suggest that ZC88 is a voltage-dependent, subtype-selective Cav2.2 channel inhibitor and can achieve an improved therapeutic window over the relatively state-independent Cav2.2-selective inhibitor, which may have potential to be developed into a novel analgesic agent.

A Novel Explanation for Observed CaMKII Dynamics in Dendritic Spines with Added EGTA or BAPTA

We present a simplified reaction network in a single well-mixed volume that captures the general features of CaMKII dynamics observed during both synaptic input and spine depolarization. Our model can also account for the greater-than-control CaMKII activation observed with added EGTA during depolarization. Calcium input currents are modeled after experimental observations, and existing models of calmodulin and CaMKII autophosphorylation are used. After calibration against CaMKII activation data in the absence of chelators, CaMKII activation dynamics due to synaptic input via n-methyl-d-aspartate receptors are qualitatively accounted for in the presence of the chelators EGTA and BAPTA without additional adjustments to the model. To account for CaMKII activation dynamics during spine depolarization with added EGTA or BAPTA, the model invokes the modulation of CaV2.3 (R-type) voltage-dependent calcium channel (VDCC) currents observed in the presence of EGTA or BAPTA. To our knowledge, this is a novel explanation for the increased CaMKII activation seen in dendritic spines with added EGTA, and suggests that differential modulation of VDCCs by EGTA and BAPTA offers an alternative or complementary explanation for other experimental results in which addition of EGTA or BAPTA produces different effects. Our results also show that a simplified reaction network in a single, well-mixed compartment is sufficient to account for the general features of observed CaMKII dynamics.

Simulated microgravity inhibits L-type calcium channel currents partially by the up-regulation of miR-103 in MC3T3-E1 osteoblasts.

L-type voltage-sensitive calcium channels (LTCCs), particularly Cav1.2 LTCCs, play fundamental roles in cellular responses to mechanical stimuli in osteoblasts. Numerous studies have shown that mechanical loading promotes bone formation, whereas the removal of this stimulus under microgravity conditions results in a reduction in bone mass. However, whether microgravity exerts an influence on LTCCs in osteoblasts and whether this influence is a possible mechanism underlying the observed bone loss remain unclear. In the present study, we demonstrated that simulated microgravity substantially inhibited LTCC currents and suppressed Cav1.2 at the protein level in MC3T3-E1 osteoblast-like cells. In addition, reduced Cav1.2 protein levels decreased LTCC currents in MC3T3-E1 cells. Moreover, simulated microgravity increased miR-103 expression. Cav1.2 expression and LTCC current densities both significantly increased in cells that were transfected with a miR-103 inhibitor under mechanical unloading conditions. These results suggest that simulated microgravity substantially inhibits LTCC currents in osteoblasts by suppressing Cav1.2 expression. Furthermore, the down-regulation of Cav1.2 expression and the inhibition of LTCCs caused by mechanical unloading in osteoblasts are partially due to miR-103 up-regulation. Our study provides a novel mechanism for microgravity-induced detrimental effects on osteoblasts, offering a new avenue to further investigate the bone loss induced by microgravity.

Presynaptic nanodomains: a tale of two synapses

Here we summarize the evidence from two “giant” presynaptic terminals—the squid giant synapse and the mammalian calyx of Held—supporting the involvement of nanodomain calcium signals in triggering of neurotransmitter release. At the squid synapse, there are three main lines of experimental evidence for nanodomain signaling. First, changing the size of the unitary calcium channel current by altering external calcium concentration causes a non-linear change in transmitter release, while changing the number of open channels by broadening the presynaptic action potential causes a linear change in release. Second, low-affinity calcium indicators, calcium chelators, and uncaging of calcium all suggest that presynaptic calcium concentrations are as high as hundreds of micromolar, which is more compatible with a nanodomain type of calcium signal. Finally, neurotransmitter release is much less affected by the slow calcium chelator, ethylene glycol tetraacetic acid (EGTA), in comparison to the rapid chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Similarly, as the calyx of Held synapse matures, EGTA becomes less effective in attenuating transmitter release while the number of calcium channels required to trigger a single fusion event declines. This suggests a developmental transformation of microdomain to nanodomain coupling between calcium channels and transmitter release. Calcium imaging and uncaging experiments, in combination with simulations of calcium diffusion, indicate the peak calcium concentration seen by presynaptic calcium sensors reaches at least tens of micromolar at the calyx of Held. Taken together, data from these provide a compelling argument that nanodomain calcium signaling gates very rapid transmitter release.

Effect of gingerol on colonic motilityviainhibition of calcium channel currents in rats

To investigate the effect of gingerol on colonic motility and the action of L-type calcium channel currents in this process. The distal colon was cut along the mesenteric border and cleaned with Ca(2+)-free physiological saline solution. Muscle strips were removed and placed in Ca(2+)-free physiological saline solution, which was oxygenated continuously. Longitudinal smooth muscle samples were prepared by cutting along the muscle strips and were then placed in a chamber. Mechanical contractile activities of isolated colonic segments in rats were recorded by a 4-channel physiograph. Colon smooth muscle cells were dissociated by enzymatic digestion. L-type calcium currents were recorded using the conventional whole-cell patch-clamp technique. Gingerol inhibited the spontaneous contraction of colonic longitudinal smooth muscle in a dose-dependent manner with inhibition percentages of 13.3% ± 4.1%, 43.4% ± 3.9%, 78.2% ± 3.6% and 80.5% ± 4.5% at 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L, respectively (P < 0.01). Nifedipine, an L-type calcium channel blocker, diminished the inhibition of colonic motility by gingerol. Gingerol inhibited L-type calcium channel currents in colonic longitudinal myocytes of rats. At a 75 μmol/L concentration of gingerol, the percentage of gingerol-induced inhibition was diminished by nifedipine from 77.1% ± 4.2% to 42.6% ± 3.6% (P < 0.01). Gingerol suppressed IBa in a dose-dependent manner, and the inhibition rates were 22.7% ± 2.38%, 35.77% ± 3.14%, 49.78% ± 3.48% and 53.78% ± 4.16% of control at 0 mV, respectively, at concentrations of 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L (P < 0.01). The steady-state activation curve was shifted to the right by treatment with gingerol. The value of half activation was -14.23 ± 1.12 mV in the control group and -10.56 ± 1.04 mV in the 75 μmol/L group (P < 0.05) with slope factors, Ks, of 7.16 ± 0.84 and 7.02 ± 0.93 (P < 0.05) in the control and 75 μmol/L groups, respectively. However, the steady-state inactivation curve was not changed, with a half-inactivation voltage, 0.5 V, of -27.43 ± 1.26 mV in the control group and -26.56 ± 1.53 mV in the 75 μmol/L gingerol group (P > 0.05), and a slope factor, K, of 13.24 ± 1.62 in the control group and 13.45 ± 1.68 (P > 0.05) in the 75 μmol/L gingerol group. Gingerol inhibits colonic motility by preventing Ca(2+) influx through L-type calcium channels.

Effects of aging in Parkinson�s disease: Role of Ca channel in dopamine neuron computational model

Effects of aging in Parkinson�s disease: Role of Ca channel in dopamine neuron computational model

Leukotriene B4 Inhibits L-Type Calcium Channels via p38 Signaling Pathway in Vascular Smooth Muscle Cells

Background/Aims: Arachidonic acid (AA) and its metabolites are important endogenous lipid messengers. In this study, we test the effect of Leukotriene B₄ (LTB₄), a 5-lipoxygenase metabolite of AA, on L-type calcium channels in A7r5 rat aortic vascular smooth muscle cells. Methods: L-type calcium channel currents were recorded by a patch-clamp technique. The mRNA expression of Ca_V1.2 was determined by Real-time RT-PCR. The protein expression of Ca_V1.2 and p38 activity was determined by Western blot analysis. Results: LTB₄ inhibits L-type channel currents in A7r5 cells in a dose-and time- dependent manner. LTB₄ reduced the mRNA/protein expression of Ca_V1.2 channels in A7r5 cells. BLT1 receptor antagonist LY29311 abrogated the inhibitory effect of LTB₄, while BLT2 receptor antagonist LY255283 had no effect. 5Z-7-oxozeaenol and SB203580, which block TAK1 and p38 kinase respectively, abrogated the LTB₄ inhibitory effect on L-type calcium channels. LTB₄ increased p38 activity in A7r5 cells. Blockage of Src, PI3K, JNK and NF-κB kinase had no effects on LTB₄ inhibition of L-type calcium channel currents in A7r5 cells. Conclusion: We conclude that LTB₄ inhibits L-type calcium channels through BLT1-TAk1-p38 signaling pathway. The LTB₄ inhibitory effect on L-type calcium channels may be involved in its pathological processes such as atherosclerosis.

Effects of aging in Parkinson�s disease: Role of L - type Ca channel in dopamine neuron computational model

Effects of aging in Parkinson�s disease: Role of L - type Ca channel in dopamine neuron computational model

Zonisamide-induced acute kidney injury.

Zonisamide is a new generation anticonvulsant indicated for the adjunctive management of partial seizures in adults [1]. The anticonvulsant activity of zonisamide is predominantly related to the inhibition of voltage-gated sodium action potentials and reduction of T-type calcium-channel currents. Because of its broad mechanism of action, zonisamide is used for many types of epilepsy and in patients whose epilepsy is resistant to other antiepileptic drugs (AEDs) [2,3]. There is insufficient evidence to demonstrate the superior clinical effectiveness of zonisamide compared to other adjunctive AEDs; however, its long half-life of 63 h supports convenient once- or twice-daily dosing. In vitro studies have demonstrated antioxidant and neuroprotective properties at therapeutic doses, suggesting that zonisamide may protect against ischemic damage and recurrent seizure activity [4]. Therapy is generally well tolerated; the most common reported adverse events include somnolence in 17% of patients and dizziness and anorexia in 13% of patients. Zonisamide may be an alternative in some patients over agents associated with weight gain, such as valproate or pregabalin [5,6]. Zonisamide is metabolized by acetylation to N-acetyl zonisamide and reduction to 2-sulfamoylacetyl phenol (SMAP) by cytochrome P450 isoenzyme 3A4. Zonisamide primarily undergoes renal excretion with 62% recovered from urine and 3% from feces following multiple doses [1,7]. The package insert was recently updated to reflect a risk of hyperchloremic, nonanion gap metabolic acidosis due to weak inhibition of carbonic anhydrase and increased renal excretion of bicarbonate. Furthermore, according to the manufacturer's label, zonisamide is not recommended for use in patients with renal failure (defined by the manufacturer as an estimated GFR < 50 mL/min); use of this agent in patients with renal insufficiency, defined as having a GFR < 60 mL/min according to the National Kidney Foundation's Kidney Disease Outcomes Quality Initiative, requires slower titration and more frequent clinical monitoring for toxicities and adverse effects. Avoidance of use of zonisamide in individuals with end-stage kidney disease (e.g., GFR < 15 mL/min or on dialysis) is prudent based on manufacturer recommendations. Zonisamide has been associated with a statistically significant 8% mean increase in serum creatinine and blood urea nitrogen in clinical trials; however, this effect dissipated after therapy was discontinued. Of note, a case report has demonstrated hypersensitivity syndrome with acute kidney injury due to zonisamide; however; acute kidney injury has not been previously reported as an adverse event of zonisamide [7]. We present a case report that suggests that such an association exists as demonstrated by acute kidney injury in a patient on two separate occasions.

Dicarba analogues of α-conotoxin RgIA. Structure, stability, and activity at potential pain targets.

α-Conotoxin RgIA is both an antagonist of the α9α10 nicotinic acetylcholine receptor (nAChR) subtype and an inhibitor of high-voltage-activated N-type calcium channel currents. RgIA has therapeutic potential for the treatment of pain, but reduction of the disulfide bond framework under physiological conditions represents a potential liability for clinical applications. We synthesized four RgIA analogues that replaced native disulfide pairs with nonreducible dicarba bridges. Solution structures were determined by NMR, activity assessed against biological targets, and stability evaluated in human serum. [3,12]-Dicarba analogues retained inhibition of ACh-evoked currents at α9α10 nAChRs but not N-type calcium channel currents, whereas [2,8]-dicarba analogues displayed the opposite pattern of selectivity. The [2,8]-dicarba RgIA analogues were effective in HEK293 cells stably expressing human Cav2.2 channels and transfected with human GABAB receptors. The analogues also exhibited improved serum stability over the native peptide. These selectively acting dicarba analogues may represent mechanistic probes to explore analgesia-related biological receptors.

The effects of vasoactive peptide urocortin 2 on hemodynamics in spontaneous hypertensive rat and the role of L-type calcium channel and CRFR2

BackgroundUrocortin (UCN) is a newly identified vascular-active peptide that has been shown to reverse cardiovascular remodeling and improve left ventricular (LV) function. The effects and mechanism of urocortin 2 (UCN2) in vivo on the electrical remodeling of left ventricle and the hemodynamics of hypertensive objectives have not been investigated. MethodsUCN2 (1μg/kg/d, 3.5μg/kg/d or 7μg/kg/d) was intravenously injected for 2 weeks and its effects on hemodynamics in spontaneously hypertensive rats (SHRs) observed. The whole-cell patch clamp technique was used to explore the effects of UCN2 on the electrical remodeling of left ventricular cardiomyocytes. The flow cytometry method was used to determine the content of fluorescence calcium in myocardium. ResultsUCN2 improved the systolic and diastolic function of SHRs as demonstrated by decreased left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), increased +dp/dtmax and −dp/dtmax and decreased cAMP level. UCN2 inhibited the opening of L-type calcium channel and decreased the calcium channel current of cardiomyocytes. In addition, UCN2 also decreased the contents of fluorescence calcium in SHR myocardium. However, astressin2-B (AST-2B), the antagonist of corticotropin-releasing factor receptor 2 (CRFR2), could reverse the inhibitory effects of UCN2 on calcium channel. ConclusionUCN2 can modulate electrical remodeling of the myocardium and hemodynamics in an experimental model of SHR via inhibition of L-type calcium channel and CRFR2 in cardiomyocytes.

Somatostatin receptor subtype 4 modulates L-type calcium channels via Gβγ and PKC signaling in rat retinal ganglion cells

Somatostatin subtype-4 receptors (sst4) inhibit L-type calcium channel currents (ICa) in retinal ganglion cells (RGCs). Here we identify the signaling pathways involved in sst4 stimulation leading to suppression of ICa in RGCs. Whole cell patch clamp recordings were made on isolated immunopanned RGCs using barium as a charge carrier to isolate ICa. Application of the selective sst4 agonist, L-803 (10 nM), reduced ICa by 41.2%. Pretreatment of cells with pertussis toxin (Gi/o inhibitor) did not prevent the action of L-803, which reduced ICa by 34.7%. To determine the involvement of Gβγ subunits after sst4 activation, depolarizing pre-pulse facilitation paradigms were used to remove voltage-dependent inhibition of calcium channels. Pre-pulse facilitation did not reverse the inhibitory effects of L-803 on ICa (8.4 vs. 8.8% reductions, ctrl vs. L-803); however, pharmacologic inhibition of Gβγ reduced ICa suppression by L-803 (23.0%, P < 0.05). Inhibition of PKC (GF109203X; GFX) showed a concentration-dependent effect in preventing the action of L-803 on ICa (1 μM GFX, 34.3%; 5 μM GFX, 14.6%, P < 0.05). When both PKC and Gβγ were inhibited, the effects of L-803 on ICa were blocked (1.8%, P < 0.05). These results suggest that sst4 stimulation modulates RGC calcium channels via Gβγ and PKC activation. Since reducing intracellular Ca2+ is known to be neuroprotective in RGCs, modulating these sst4 signaling pathways may provide insights to the discovery of unique therapeutic targets to reduce intracellular Ca2+ levels in RGCs.

Butanol isomers exert distinct effects on voltage-gated calcium channel currents and thus catecholamine secretion in adrenal chromaffin cells.

Butanol (C4H10OH) has been used both to dissect the molecular targets of alcohols/general anesthetics and to implicate phospholipase D (PLD) signaling in a variety of cellular functions including neurotransmitter and hormone exocytosis. Like other primary alcohols, 1-butanol is a substrate for PLD and thereby disrupts formation of the intracellular signaling lipid phosphatidic acid. Because secondary and tertiary butanols do not undergo this transphosphatidylation, they have been used as controls for 1-butanol to implicate PLD signaling. Recently, selective pharmacological inhibitors of PLD have been developed and, in some cases, fail to block cellular functions previously ascribed to PLD using primary alcohols. For example, exocytosis of insulin and degranulation of mast cells are blocked by primary alcohols, but not by the PLD inhibitor FIPI. In this study we show that 1-butanol reduces catecholamine secretion from adrenal chromaffin cells to a much greater extent than tert-butanol, and that the PLD inhibitor VU0155056 has no effect. Using fluorescent imaging we show the effect of these drugs on depolarization-evoked calcium entry parallel those on secretion. Patch-clamp electrophysiology confirmed the peak amplitude of voltage-gated calcium channel currents (ICa) is inhibited by 1-butanol, with little or no block by secondary or tert-butanol. Detailed comparison shows for the first time that the different butanol isomers exert distinct, and sometimes opposing, effects on the voltage-dependence and gating kinetics of ICa. We discuss these data with regard to PLD signaling in cellular physiology and the molecular targets of general anesthetics.

The specific study of voltage-dependent calcium channels currents in rats cerebral arteries myocytes

Objective To study the electrophysiology and pharmacology of voltage-dependent calcium channels (VDCCs) currents in cerebral artery myocytes of rats specifically to help for related arterial physiology or pathology research.Methods Myocytes were isolated enzymatically and patch clamp was used to test the resting potential and VDCCs currems in whole model,as well as effects of extracellular divalent cations concentration and Nifedipine on VDCCs currents.Results The resting potential was (50.42 ±0.26) mV,step protocol at 10 mV and ramp protocol at (9.80±0.92) mmol/L elicited maximum VDCCs currents density in (-5.22±0.51)pA/pF and (-4.89 ±0.65) pA/pF (10 mmol/L BaCl2).The maximum VDCCs currents consisted of high-voltage activation (HVA) VDCCs exclusively (P =0.17),however,denying the existence of low-voltage activation (LVA) VDCCs currents.Furthermore,the VDCCs currents density correlated with extracellular Ba2+ concentration positively (P ＜ 0.05),and of the VDCCs currents,Nifedipine was able to inhibit 86.13 0.76％ maximally with the IC50 6.02 mmol/L Conclusions Our study confirmed that HVA-VDCCs accounted for all the VDCCs currents and Nifedipine-insensitive calcium currents (NICCs) in cerebral artery myocytes of rats.Accordingly,further studies in term of ion channel (s) accounting for NICCs and its significance for myogenic tone of cerebral artery and cerebral autoregulation are required. Key words: Cerebral arteries; Voltage-dependent calcium channels; Patch clamp

Calcium channels for endocytosis.

Calcium channels for endocytosis.

Analysis of the long-term actions of gabapentin and pregabalin in dorsal root ganglia and substantia gelatinosa.

The α2δ-ligands pregabalin (PGB) and gabapentin (GBP) are used to treat neuropathic pain. We used whole cell recording to study their long-term effects on substantia gelatinosa and dorsal root ganglion (DRG) neurons. Spinal cord slices were prepared from embryonic day 13 rat embryos and maintained in organotypic culture for >5 wk (neuronal age equivalent to young adult rats). Exposure of similarly aged DRG neurons (dissociated and cultured from postnatal day 19 rats) to GBP or PGB for 5-6 days attenuated high-voltage-activated calcium channel currents (HVA ICa). Strong effects were seen in medium-sized and in small isolectin B4-negative (IB4-) DRG neurons, whereas large neurons and small neurons that bound isolectin B4 (IB4+) were hardly affected. GBP (100 μM) or PGB (10 μM) were less effective than 20 μM Mn(2+) in suppression of HVA ICa in small DRG neurons. By contrast, 5-6 days of exposure to these α2δ-ligands was more effective than 20 μM Mn(2+) in reducing spontaneous excitatory postsynaptic currents at synapses in substantia gelatinosa. Spinal actions of gabapentinoids cannot therefore be ascribed to decreased expression of HVA Ca(2+) channels in primary afferent nerve terminals. In substantia gelatinosa, 5-6 days of exposure to PGB was more effective in inhibiting excitatory synaptic drive to putative excitatory neurons than to putative inhibitory neurons. Although spontaneous inhibitory postsynaptic currents were also attenuated, the overall long-term effect of α2δ-ligands was to decrease network excitability as monitored by confocal Ca(2+) imaging. We suggest that selective actions of α2δ-ligands on populations of DRG neurons may predict their selective attenuation of excitatory transmission onto excitatory vs. inhibitory neurons in substantia gelatinosa.

Aging Decreases L-Type Calcium Channel Currents and Pacemaker Firing Fidelity in Substantia Nigra Dopamine Neurons

Substantia nigra dopamine neurons are involved in behavioral processes that include cognition, reward learning, and voluntary movement. Selective deterioration of these neurons is responsible for the motor deficits associated with Parkinson's disease (PD). Aging is the leading risk factor for PD, suggesting that adaptations occurring in dopamine neurons during normal aging may predispose individuals to the development of PD. Previous studies suggest that the unique set of ion conductances that drive spontaneous, rhythmic firing of action potentials could predispose substantia nigra dopamine neurons to selective neurodegeneration. Here we show, using patch-clamp electrophysiological recordings in brain slices, that substantia nigra dopamine neurons from mice 25-30 months of age (old) have comparable membrane capacitance and input resistance to neurons from mice 2-7 months of age (young). However, neurons from old mice exhibit slower firing rates, narrower spike widths, and more variable interspike intervals compared with neurons from young mice. Dopamine neurons from old mice also exhibit smaller L-type calcium channel currents, providing a plausible mechanism that likely contributes to the changes in impulse activity. Age-related decrements in the physiological function of dopamine neurons could contribute to the decrease in voluntary movement and other dopamine-mediated behaviors observed in aging populations. Furthermore, as pharmacological antagonism of L-type calcium channels has been proposed as a potential treatment for the early stages of PD, our results could point to a limited temporal window of opportunity for this therapeutic intervention.