HVA Currents Research Articles

The Kelch‐Like 1 Actin Binding Protein Modulates Neuronal Voltage Gated Calcium Channels

Kelch‐like 1 (KLHL1) is a neuronal actin‐binding protein first identified in humans as part of the genetic locus of the neurological movement disorder Spinocerebellar Ataxia type 8. KLHL1 is expressed in the cytosol, axons and dendrites. KLHL1 KO causes loss of post‐synaptic structures, loss of motor coordination and gait abnormalities. KLHL1 is an ion channel modulator and upregulates HVA and LVA calcium currents in vitro by increased recycling endosome activity and by alteration of the kinetics of τdeactivation. We analyzed the endogenous HVA and LVA calcium currents in neurons from WT and KLHL1 KO mice, and compared these data with the acute down‐regulation of KLHL1 using a knock‐down shRNA (KD) strategy. KLHL1 KD caused decreased HVA and LVA calcium current expression. In contrast, the KO had no differences in total LVA current density, yet changes in current kinetics suggest there is a down‐regulation of α1H and compensatory expression of α1I. HVA current density was also decreased in KO (40%) at 4–6 DIV but not at 7–10 DIV, suggesting this deficit was also compensated for after 7 DIV. Our data confirm that KLHL1 modulates LVA and HVA channel function in vivo; the compensatory modulation of currents in the KO may contribute to the mild motor phenotype seen in these mice.This material is based upon work supported by the National Science Foundation under Grant No. 1022075

Calcium-dependent inhibition of T-type calcium channels by TRPV1 activation in rat sensory neurons

We studied the inhibitory effects of transient receptor potential vanilloid-1 (TRPV1) activation by capsaicin on low-voltage-activated (LVA, T-type) Ca(2+) channel and high-voltage-activated (HVA; L, N, P/Q, R) currents in rat DRG sensory neurons, as a potential mechanism underlying capsaicin-induced analgesia. T-type and HVA currents were elicited in whole-cell clamped DRG neurons using ramp commands applied before and after 30-s exposures to 1μM capsaicin. T-type currents were estimated at the first peak of the I-V characteristics and HVA at the second peak, occurring at more positive potentials. Small and medium-sized DRG neurons responded to capsaicin producing transient inward currents of variable amplitudes, mainly carried by Ca(2+). In those cells responding to capsaicin with a large Ca(2+) influx (59% of the total), a marked inhibition of both T-type and HVA Ca(2+) currents was observed. The percentage of T-type and HVA channel inhibition was prevented by replacing Ca(2+) with Ba(2+) during capsaicin application or applying high doses of intracellular BAPTA (20mM), suggesting that TRPV1-mediated inhibition of T-type and HVA channels is Ca(2+)-dependent and likely confined to membrane nano-microdomains. Our data are consistent with the idea that TRPV1-induced analgesia may derive from indirect inhibition of both T-type and HVA channels which, in turn, would reduce the threshold of nociceptive signals generation (T-type channel inhibition) and nociceptive synaptic transmission (HVA-channels inhibition).

Extrinsic regulation of T‐type Ca2+ channel expression in chick nodose ganglion neurons

Functional expression of T-type Ca(2+) channels is developmentally regulated in chick nodose neurons. In this study we have tested the hypothesis that extrinsic factors regulate the expression of T-type Ca(2+) channels in vitro. Voltage-gated Ca(2+) currents were measured using whole-cell patch clamp recordings in E7 nodose neurons cultured under various conditions. Culture of E7 nodose neurons for 48 h with a heart extract induced the expression of T-type Ca(2+) channels without any significant effect on HVA currents. T-type Ca(2+) channel expression was not stimulated by survival promoting factors such as BDNF. The stimulatory effect of heart extract was mediated by a heat-labile, trypsin-sensitive factor. Various hematopoietic cytokines including CNTF and LIF mimic the stimulatory effect of heart extract on T-type Ca(2+) channel expression. The stimulatory effect of heart extract and CNTF requires at least 12 h continuous exposure to reach maximal expression and is not altered by culture of nodose neurons with the protein synthesis inhibitor anisomycin, suggesting that T-type Ca(2+) channel expression is regulated by a posttranslational mechanism. Disruption of the Golgi apparatus with brefeldin-A inhibits the stimulatory effect of heart extract and CNTF suggesting that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Heart extract- or CNTF-evoked stimulation of T-type Ca(2+) channel expression is blocked by the Jak/STAT and MAP kinase blockers, AG490 and U0126, respectively. This study provides new insights into the electrical differentiation of placode-derived sensory neurons and the role of extrinsic factors in regulating the functional expression of Ca(2+) channels.

Postnatal Development Differentially Affects Voltage-Activated Calcium Currents in Respiratory Rhythmic Versus Nonrhythmic Neurons of the Pre-Bötzinger Complex

The mammalian respiratory network reorganizes during early postnatal life. We characterized the postnatal developmental changes of calcium currents in neurons of the pre-Bötzinger complex (pBC), the presumed site for respiratory rhythm generation. The pBC contains not only respiratory rhythmic (R) but also nonrhythmic neurons (nR). Both types of neurons express low- and high-voltage-activated (LVA and HVA) calcium currents. This raises the interesting issue: do calcium currents of the two co-localized neuron types have similar developmental profiles? To address this issue, we used the whole cell patch-clamp technique to compare in transverse slices of mice LVA and HVA calcium current amplitudes of the two neuron populations (R and nR) during the first and second postnatal week (P0-P16). The amplitude of HVA currents did not significantly change in R pBC-neurons (P0-P16), but it significantly increased in nR pBC-neurons during P8-P16. The dehydropyridine (DHP)-sensitive current amplitudes did not significantly change during the early postnatal development, suggesting that the observed amplitude changes in nR pBC-neurons are caused by (DHP) insensitive calcium currents. The ratio between HVA calcium current amplitudes dramatically changed during early postnatal development: At P0-P3, current amplitudes were significantly larger in R pBC-neurons, whereas at P8-P16, current amplitudes were significantly larger in nR pBC-neurons. Our results suggest that calcium currents in pBC neurons are differentially altered during postnatal development and that R pBC-neurons have fully expressed calcium currents early during postnatal development. This may be critical for stable respiratory rhythm generation in the underlying rhythm generating network.

Voltage-gated calcium channels in adult rat inferior colliculus neurons

The inferior colliculus (IC) plays a key role in the processing of auditory information and is thought to be an important site for genesis of wild running seizures that evolve into tonic–clonic seizures. IC neurons are known to have Ca 2+ channels but neither their types nor their pharmacological properties have been as yet characterized. Here, we report on biophysical and pharmacological properties of Ca 2+ channel currents in acutely dissociated neurons of adult rat IC, using electrophysiological and molecular techniques. Ca 2+ channels were activated by depolarizing pulses from a holding potential of −90 mV in 10 mV increments using 5 mM barium (Ba 2+) as the charge carrier. Both low (T-type, VA) and high (HVA) threshold Ca 2+ channel currents that could be blocked by 50 μM cadmium, were recorded. Pharmacological dissection of HVA currents showed that nifedipine (10 μM, L-type channel blocker), ω-conotoxin GVIA (1 μM, N-type channel blocker), and ω-agatoxin TK (30 nM, P-type channel blocker) partially suppressed the current by 21%, 29% and 22%, respectively. Since at higher concentration (200 nM) ω-agatoxin TK also blocks Q-type channels, the data suggest that Q-type Ca 2+ channels carry approximately 16% of HVA current. The fraction of current (approximately 12%) resistant to the above blockers, which was blocked by 30 μM nickel and inactivated with τ of 15–50 ms, was considered as R-type Ca 2+ channel current. Consistent with the pharmacological evidences, Western blot analysis using selective Ca 2+ channel antibodies showed that IC neurons express Ca 2+ channel α 1A, α 1B, α 1C, α 1D, and α 1E subunits. We conclude that IC neurons express functionally all members of HVA Ca 2+ channels, but only a subset of these neurons appear to have developed functional LVA channels.

Farnesol modulates membrane currents in human retinal glial cells.

Farnesol, a C(15) natural isoprenoid, exerts complex modulating effects on the membrane permeability of human retinal glial (Müller) cells. Several glial cationic currents were examined. At low micromolar concentrations, farnesol reduced the amplitudes of all fast and depolarization-activated membrane currents expressed by Müller cells, that is, currents through 1) transient low-voltage-activated (LVA; IC(50) = 2.2 microM), 2) sustained high-voltage-activated Ca(2+) channels (HVA; IC(50) = 1.2 microM), 3) fast Na(+) channels (IC(50) = 9.0 microM), and 4) transient (A-type) K(+) channels (IC(50) = 4.7 microM). Furthermore, farnesol shifted the activation of LVA and HVA currents to more depolarized potentials by 21.3 +/- 7.4 mV and 8.3 +/- 4.5 mV, respectively. On the other hand, neither inwardly rectifying nor iberiotoxin-sensitive calcium-activated K(+) currents were affected by farnesol. Therefore, farnesol is assumed to be a biologically active substance that regulates ion channel activity in the glial cell membrane. Depressing rapid changes of the membrane potential and supporting a stable hyperpolarized status of the glial cells may enhance the efficiency of crucial glial functions such as extracellular K(+) clearance and neurotransmitter uptake.

Calcium currents in acutely isolated stellate and pyramidal neurons of rat entorhinal cortex

Calcium currents were studied in morphologically identified pyramidal and stellate neurons acutely isolated from layer II/III of rat entorhinal cortex, using the whole-cell patch-clamp configuration. The peak amplitude of high-voltage activated current (HVA) measured at +10 mV was not different in both neuron populations with 0.94±0.08 nA for pyramidal and 1.03±0.08 nA for stellate cells. Stellate neurons had a larger capacitance (14.4±1.1 pF) than pyramidal neurons (9.6±0.8 pF), indicating a 50% larger cell surface. Most striking was the difference between the current density in stellate (79±8 pA/pF) versus pyramidal neurons (113±13 pA/pF). The potential of half maximal inactivation was not different: −37±2 mV (pyramidals) and −37±3 mV (stellates). Half of the cells contained a low-voltage activated calcium current (LVA) with a peak amplitude that was twice as large in stellate as in pyramidal neurons (0.21±0.04 nA resp. 0.11±0.03 nA; at −50 mV). In contrast to the HVA component, the current density of the LVA component was not different between cell types (13±3 pA/pF vs. 13±2 pA/pF). This implies that the relative abundance of LVA and HVA currents in stellate and pyramidal neurons is different which could result in different firing characteristics. The potential of half maximal LVA inactivation was −88±4 mV (pyramidals) and −85±3 mV (stellates). The slope of the voltage dependent steady state inactivation was steeper in stellate (7±1 mV) than in pyramidal cells (10±2 mV).

Differential sensitivity to intracellular pH among high- and low-threshold Ca2+ currents in isolated rat CA1 neurons.

The effects of intracellular pH (pHi) on high-threshold (HVA) and low-threshold (LVA) calcium currents were examined in acutely dissociated rat hippocampal Ca1 neurons with the use of the whole cell patch-clamp technique (21-23 degrees C). Internal pH was manipulated by external exposure to the weak base NH4Cl or in some cases to the weak acid Na-acetate (20 mM) at constant extracellular pH (7.4). Confocal fluorescence measurements using the pH-sensitive dye SNARF-1 in both dialyzed and intact cells confirmed that NH4Cl caused a reversible alkaline shift. However, the external TEA-Cl concentration used during ICa recording was sufficient to abolish cellular acidification upon NH4Cl wash out. With 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the pipette, NH4Cl exposure reversibly enhanced HVA currents by 29%, whereas exposure to Na-acetate markedly and reversibly depressed HVA Ca currents by 62%. The degree to which NH4Cl enhanced HVA currents was inversely related to the internal HEPES concentration but was unaffected when internal ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was replaced by equimolar bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). When depolarizing test pulses were applied shortly after break-in (Vh = -100 mV), NH4Cl caused a proportionally greater increase in the sustained current relative to the peak. The dihydropyridine Ca channel antagonist nifedipine (5 microM) blocked nearly all of this sustained current. A slowly inactivating nifedipine-sensitive (L-type) HVA current could be evoked from a depolarized holding potential of -50 mV; NH4Cl enhanced this current by 40 +/- 3% (mean +/- SE) and reversibly shifted the tail-current activation curve by +6-8 mV. L-type currents exhibited more rapid rundown than N-type currents; HVA currents remaining after prolonged cell dialysis, or in the presence of nifedipine, inactivated rapidly and were depressed by omega-conotoxin (GVIA). NH4Cl enhanced these N-type currents by 76 +/- 9%. LVA Ca currents were observed in 32% of the cells and exhibited little if any rundown. These amiloride-sensitive currents activated at voltages negative to -50 mV, were enhanced by extracellular alkalosis and depressed by extracellular acidosis, but were unaffected by exposure to either NH4Cl or NaAC. These results demonstrate that HVA Ca currents in hippocampal CA1 neurons are bidirectionally modulated by internal pH shifts, and that N-type currents are more sensitive to alkaline shifts than are L- or T-type (N > L > T). Our findings strengthen the idea that distinct cellular processes governed by different Ca channels may be subject to selective modulation by uniform shifts in cytosolic pH.

Voltage gated calcium channels in molluscs: classification, Ca2+ dependent inactivation, modulation and functional roles.

Molluscan neurons and muscle cells express transient (T-type like) and sustained LVA calcium channels, as well as transient and sustained HVA channels. In addition weakly voltage sensitive calcium channels are observed. In a number of cases toxin or dihydropyridine sensitivity justifies classification of the HVA currents in L, N or P-type categories. In many cases, however, pharmacological characterization is still preliminary. Characterization of novel toxins from molluscivorous Conus snails may facilitate classification of molluscan calcium channels. Molluscan preparations have been very useful to study calcium dependent inactivation of calcium channels. Proposed mechanisms explain calcium dependent inactivation through direct interaction of Ca2+ with the channel, through dephosphorylation by calcium dependent phosphatases or through calcium dependent disruption of connections with the cytoskeleton. Transmitter modulation operating through various second messenger mediated pathways is well documented. In general, phosphorylation through PKA, cGMP dependent PK or PKC facilitates the calcium channels, while putative direct G-protein action inhibits the channels. Ca2+ and cGMP may inhibit the channels through activation of phosphodiesterases or phosphatases. Detailed evidence has been provided on the role of sustained LVA channels in pacemaking and the generation of firing patterns, and on the role of HVA channels in the dynamic changes in action potentials during spiking, the regulation of the release of transmitters and hormones, and the regulation of growth cone behavior and neurite outgrowth. The accessibility of molluscan preparations (e.g. the squid giant synapse for excitation release studies, Helisoma B5 neuron for neurite and synapse formation) and the large body of knowledge on electrophysiological properties and functional connections of identified molluscan neurons (e.g. sensory neurons, R15, egg laying hormone producing cells, etc.) creates valuable opportunities to increase the insight into the functional roles of calcium channels.

Ca2+ currents in cockroach neurones: properties and modulation by neurohormone D.

In dorsal unpaired median (DUM) neurones isolated from the terminal ganglion of the cockroach Periplaneta americana two types of Ca2+ currents were found: a transient one activated by lower depolarizations (LVA) and a sustained one activated by higher voltage jumps (HVA). Both currents were strongly reduced by 100 microM Cd2+; Ni2+ was less effective. The HVA currents were insensitive to dihydropyridines, but sensitive to phenylalkylamines. The LVA component was sensitive to omega-conotoxin GVIA and SK&F96365 whereas the HVA component was insensitive to both blockers. The octapeptide neurohormone D (NHD), a member of the family of adipokinetic hormones (AKH-peptides), is known to increase the spiking frequence in these DUM cells. We demonstrate that NHD increases the LVA current component and decreases the HVA current component. These effects of NHD can explain the previously found NHD-induced stimulation of Ca2+ dependent K+ currents.

Tetrandrine effects on calcium currents in cultured neurones of foetal mice

Tetrandrine, a bis-benzyl-isoquinoline alkaloid from the roots of Stefania Tetranda blocks calcium currents in cultured neurones of the spinal cord of foetal mice. The concentration of half-maximal effect (Kd) measured 100 ms after the beginning of a depolarizing pulse (-80 mV-0 mV) is 8 microM. While high- and low-voltage activated (HVA, LVA) currents are both blocked by Tetrandrine, the slowly inactivating HVA currents appear to be a primary target.

Differential expression of voltage-gated Ca 2+-currents in cultivated aortic myocytes

The expression of different types of Ca 2+-channels was studied using the whole-cell patch-clamp technique in cultured rat aortic smooth-muscle myocytes. Ca 2+-currents were identified as either low- or high voltage-activated ( I Ca,LVA or I Ca,HVA, respectively) based on their distinct voltage-dependences of activation and inactivation, decay kinetics using Ba 2+ as the charge carrier and sensitivity to dihydropyridines. The heterogeneity in the functional expression of the two types of Ca 2+-channels in the cultured myocytes delineated four distinct phenotypes; (i), cells exhibiting only LVA currents; (ii), cells exhibiting only HVA currents; (iii), cells exhibiting both LVA and HVA currents and (iv), cells exhibiting no current. The myocytes exclusively expressed HVA currents both during the first five days in primary culture and after the cells had reached confluence (> 15 days). In contrast, LVA currents were expressed transiently between 5 and 15 days, during which time the cells were proliferating and had transient loss of contractility. Thus both LVA and HVA Ca 2+-current types contribute to Ca 2+-signalling in cultured rat aortic myocytes. However, the differential expression of the two Ca 2+ current types associated with differences in contractile and proliferative phenotypes suggest that they serve distinct cellular functions. Our results are consistent with the idea that LVA current expression is important for cell proliferation.

Intra and extracellular surface charges near Ca2+ channels in neurons and neuroblastoma cells.

The properties of low (LVA) and high (HVA) voltage-activated calcium currents were investigated in rat sensory neurons and a murine neuroblastoma cell line exposed to various concentrations of intra- or extracellular monovalent ([c+]i/o) and trivalent ([c3+]i/o) cations. In neurons, when [c+]i was changed from 150 to 20 mM, positive shifts of 18-28 mV were observed in activation curves of both LVA and HVA currents, as well as in LVA inactivation curves. Extracellularly, in divalent-free solutions, [c+]o of 20-50 mM produced medium (12-22 mV) negative shifts of the LVA channel properties. These data were used to estimate, by a "screening" model, a negative surface charge density around neuron's calcium channels of 1/1,000 and 1/1,325 eA-2 at the outside or inside face, respectively. In the presence of physiological concentrations of divalent cations, [c+]o of 20-60 mM caused smaller (4-11 mV) negative shifts of the activation and inactivation curves, which can be explained by assuming a partial neutralization of negative charges by divalent cations. By applying the above procedure to LVA channels of neuroblastoma cells, the ratio of extra- to intracellular surface charge density turned out to be more than tenfold higher than in neurons. Effects produced by [c3+]i/o were not in agreement with expectations based on screening or binding models.

Ca currents in human neuroblastoma IMR32 cells: kinetics, permeability and pharmacology

We have investigated the kinetics, permeability and pharmacological properties of Ca channels in in vitro differentiated IMR32 human neuroblastoma cells. The low-threshold (LVA, T) Ca current activated positive to -50 mV and inactivated fully within 100 ms in a voltage-dependent manner. This current persisted in the presence of 3.2 microM omega-conotoxin (omega-CgTx) or 40 microM Cd and showed a weaker sensitivity to Ni and amiloride than in other neurons. The high-threshold Ca currents (HVA,L and N) turned on positive to -30 mV, and inactivated slowly and incompletely during pulses of 200 ms duration. The amplitude of the HVA currents and the number of 125I-omega-CgTx binding sites increased markedly during cell differentiation. In agreement with recent reports, 6.4 microM omega-CgTx blocked only about 85% of the Ba currents through HVA channels in 50% of the cells. Residual omega-CgTx-resistant currents proved to be more sensitive to dihydropyridines (DHP) than total HVA currents. Bay K 8644 (1 microM) had a clear agonistic action on omega-CgTx-resistant currents and was preferred to other Ca antagonists for identifying HVA DHP-sensitive channels. Compared to the omega-CgTx-sensitive, the DHP-sensitive currents turned on at slightly more negative potentials and showed a weaker sensitivity to voltage. The two HVA currents were otherwise hardly distinguishable in terms of activation/inactivation kinetics, Ca/Ba permeability and sensitivity to holding potentials. This suggests that currently used criteria for identifying multiple types of neuronal Ca channels (T;L,N) may be widely misleading if not supported by pharmacological assays.