Molecular and functional characterization of a family of rat brain T-type calcium channels.

Voltage-gated calcium channels represent a heterogenous family of calcium-selective channels that can be distinguished by their molecular, electrophysiological, and pharmacological characteristics. We report here the molecular cloning and functional expression of three members of the low voltage-activated calcium channel family from rat brain (alpha(1G), alpha(1H), and alpha(1I)). Northern blot and reverse transcriptase-polymerase chain reaction analyses show alpha(1G), alpha(1H), and alpha(1I) to be expressed throughout the newborn and juvenile rat brain. In contrast, while alpha(1G) and alpha(1H) mRNA are expressed in all regions in adult rat brain, alpha(1I) mRNA expression is restricted to the striatum. Expression of alpha(1G), alpha(1H), and alpha(1I) subunits in HEK293 cells resulted in calcium currents with typical T-type channel characteristics: low voltage activation, negative steady-state inactivation, strongly voltage-dependent activation and inactivation, and slow deactivation. In addition, the direct electrophysiological comparison of alpha(1G), alpha(1H), and alpha(1I) under identical recording conditions also identified unique characteristics including activation and inactivation kinetics and permeability to divalent cations. Simulation of alpha(1G), alpha(1H), and alpha(1I) T-type channels in a thalamic neuron model cell produced unique firing patterns (burst versus tonic) typical of different brain nuclei and suggests that the three channel types make distinct contributions to neuronal physiology.

alpha 1B N-type calcium channel isoforms with distinct biophysical properties.

N-type calcium channels both generate the initial calcium signal to trigger neurotransmitter release and also interact with synaptic release proteins at many mammalian central nervous system synapses. Two isoforms of the alpha 1B N-type channel from rat brain (alpha 1B-I and alpha 1B-II) were found to differ in four regions: (1) a glutamate (Glu) to glycine (Gly) substitution in domain I S3; (2) a Gly to Glu substitution in the domain I-II linker; (3) the insertion or deletion of an alanine (Ala) in the domain I-II linker; and (4) the presence or absence of serine/phenylalanine/methionine/glycine (SFMG) in the linker between domain III S3-S4. Comparison of the electrophysiological properties of the alpha 1B-I and alpha 1B-II N-type channels shows that they exhibit distinct kinetics as well as altered current-voltage relations. Utilizing chimeric alpha 1B-I and alpha 1B-II cDNAs, we show that: (1) the Glu 177 to Gly substitution in domain I S3 increases the rate of activation by approximately 15-fold; (2) the presence or absence of Ala 415 in the domain I-II linker alters current-voltage relations by approximately 10 mV but does not affect channel kinetics; (3) the substitution of Gly 387 to Glu in the domain I-II linker also has no effect on kinetics; and (4) the presence or absence of SFMG (1236-1239) in domain III S3-S4 did not significantly affect channel current-voltage relations, kinetics, or steady state inactivation. We conclude that molecularly distinct alpha 1B isoforms are expressed in rat brain and may account for some of the functional diversity of N-type currents in native cells.

Inhibition of neuronal calcium channels by a novel peptide spider toxin, DW13.3.

Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently expressed calcium channels in oocytes showed that DW13.3 had the highest affinity for alpha1A, followed by alpha1B > alpha1C > alpha1E. The affinity of DW13.3 for alpha1B N-type currents varied by 10-fold between expressed channels and native currents. Although block occurred in a similar 1:1 manner for all subtypes, DW13.3 produced a partial block of both alpha1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand omega-conotoxin MVIIC (but not omega-agatoxin IVA) from its binding site in Purkinje neurons suggests that DW13.3 binds to a site close to the pore of the channel. The inhibition of different subtypes of calcium channels by DW13.3 reflects a common "macro" binding site present on all calcium channels except T-type.

The alpha 1E calcium channel exhibits permeation properties similar to low-voltage-activated calcium channels.

The physiological and pharmacological properties of the alpha 1E calcium (Ca) channel subtype do not exactly match any of the established categories described for native neuronal Ca currents. Many of the key diagnostic features used to assign cloned Ca channels to their native counterparts, however, are dependent on a number of factors, including cellular environment, beta subunit coexpression, and modulation by second messengers and G-proteins. Here, by examining the intrinsic pore characteristics of a family of transiently expressed neuronal Ca channels, we demonstrate that the permeation properties of alpha 1E closely resemble those described for a subset of low-threshold Ca channels. The alpha 1A (P-/Q-type), alpha 1B (N-type), and alpha 1C (L-type) high-threshold Ca channels all exhibit larger whole-cell currents with barium (Ba) as the charge carrier as compared with Ca or strontium (Sr). In contrast, macroscopic alpha 1E currents are largest in Sr, followed by Ca and then Ba. The unique permeation properties of alpha 1E are maintained at the single-channel level, are independent of the nature of the expression system, and are not affected by coexpression of alpha 2 and beta subunits. Overall, the permeation characteristics of alpha 1E are distinct from those described for R-type currents and share some similarities with native low-threshold Ca channels.

Determinants of the G protein-dependent opioid modulation of neuronal calcium channels.

The modulation of a family of cloned neuronal calcium channels by stimulation of a coexpressed mu opioid receptor was studied by transient expression in Xenopus oocytes. Activation of the morphine receptor with the synthetic enkephalin [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) resulted in a rapid inhibition of alpha1A (by approximately 20%) and alpha1B (by approximately 55%) currents while alpha1C and alpha1E currents were not significantly affected. The opioid-induced effects on alpha1A and alpha1B currents were blocked by pertussis toxin and the GTP analogue guanosine 5'-[beta-thio]diphosphate. Similar to modulation of native calcium currents, DAMGO induced a slowing of the activation kinetics and exhibited a voltage-dependent inhibition that was partially relieved by application of strong depolarizing pulses. alpha1A currents were still inhibited in the absence of coexpressed Ca channel alpha2 and beta subunits, suggesting that the response is mediated by the alpha1 subunit. Furthermore, the sensitivity of alpha1A currents to DAMGO-induced inhibition was increased approximately 3-fold in the absence of a beta subunit. Overall, the results show that the alpha1A (P/Q type) and the alpha1B (N type) calcium channels are selectively modulated by a GTP-binding protein (G protein). The results raise the possibility of competitive interactions between beta subunit and G protein binding to the alpha1 subunit, shifting gating in opposite directions. At presynaptic terminals, the G protein-dependent inhibition may result in decreased synaptic transmission and play a key role in the analgesic effect of opioids and morphine.

Determinants of PKC-dependent modulation of a family of neuronal calcium channels.

The modulation of Ca2+ channel activity by protein kinases contributes to the dynamic regulation of neuronal physiology. Using the transient expression of a family of neuronal Ca2+ channels, we have identified several factors that contribute to the PKC-dependent modulation of Ca2+ channels. First, the nature of the Ca2+ channel alpha 1 subunit protein is critical. Both alpha 1B and alpha 1E channels exhibit a 30%-40% increase in peak currents after exposure to phorbol esters, whereas neither alpha 1A nor alpha 1C channels are significantly affected. This up-regulation can be mimicked for alpha 1E channels by stimulation of a coexpressed metabotropic glutamate receptor (type 1 alpha) through a PKC-dependent pathway. Second, PKC-stimulated up-regulation is dependent upon coexpression with a Ca2+ channel beta subunit. Third, substitution of the cytoplasmic domain I-II linker from alpha 1B confers PKC sensitivity to alpha 1A channels. The results provide direct evidence for the modulation of a subset of neuronal Ca2+ channels by PKC and implicate alpha 1 and beta subunit interactions in regulating channel activity via second messenger pathways.

Long-term modulation of inward currents in O2 chemoreceptors by chronic hypoxia and cyclic AMP in vitro.

In mammals, ventilatory acclimatization to hypoxia is associated with an enhanced chemosensitivity of the O2-sensing carotid body, resulting in an increased respiratory drive. To test whether this sensitization involves long-term modulation of ion channel function in endogenous O2 chemoreceptors, i.e., type 1 cells, we exposed cultures of dissociated rat carotid body to chronic hypoxia (6% O2) for 1-2 weeks, before monitoring the electrophysiological properties of type 1 cells using whole-cell, perforated patch recording. Chronic hypoxia augmented voltage-dependent inward Na+ and Ca2+ currents in type 1 cells, without significant changes in voltage dependence of activation or steady-state inactivation. However, after normalizing for the concomitant increase in cell size, indicated by the whole-cell capacitance, only the Na+ current density was significantly enhanced. The Na+ current was sensitive to tetrodotoxin (TTX; 0.5-1 microM) or choline substitution, whereas most of the Ca2+ current was sensitive to the L-type calcium channel blocker, nifedipine (10 microM). Several of these effects of hypoxia were mimicked qualitatively by growing normoxic cultures in the presence of agents that elevate intracellular cyclic AMP, including dibutyryl cAMP (db-cAMP; 200 microM-1 mM) and forskolin (10 microM); treatment with similar concentrations of dibutyryl cyclic GMP was ineffective. Na+ channel induction by db-cAMP was abolished by the protein synthesis inhibitor, cycloheximide (90-180 microM). In current-clamp mode, these altered chemoreceptors had typical resting potentials of approximately -55 mV, and following depolarization often fired multiple spikes that appeared to consist of both short-duration Na+ and long-duration Ca2+ components. We propose that chronic hypoxia, acting in part through cAMP-dependent pathways, increases electrical excitability and calcium mobilization in type 1 cells, and these adaptations may help enhance chemosensitivity during hypoxic acclimatization.

Voltage-dependent facilitation of a neuronal alpha 1C L-type calcium channel.

Calcium entry into excitable cells through voltage-gated calcium channels can be influenced by both the rate and pattern of action potentials. We report here that a cloned neuronal alpha 1C L-type calcium channel can be facilitated by positive pre-depolarization. Both calcium and barium were effective as charge carriers in eliciting voltage-dependent facilitation. The induction of facilitation was shown to be independent of intracellular calcium levels, G-protein interaction and the level of phosphatase activity. Facilitation was reduced by the injection of inhibitors of protein kinase A and required the coexpression of a calcium channel beta subunit. In contrast, three neuronal non-L-type calcium channels, alpha 1A, alpha 1B and alpha 1E, were not subject to voltage-dependent facilitation when coexpressed with a beta subunit. The results indicate that the mechanism of neuronal L-type calcium channel facilitation involves the interaction of alpha 1 and beta subunits and is dependent on protein kinase A activity. The selective voltage-dependent modulation of L-type calcium channels is likely to play an important role in neuronal physiology and plasticity.

Localization and functional properties of a rat brain alpha 1A calcium channel reflect similarities to neuronal Q- and P-type channels.

Functional expression of the rat brain alpha 1A Ca channel was obtained by nuclear injection of an expression plasmid into Xenopus oocytes. The alpha 1A Ca current activated quickly, inactivated slowly, and showed a voltage dependence typical of high voltage-activated Ca channels. The alpha 1A current was partially blocked (approximately 23%) by omega-agatoxin IVA (200 nM) and substantially blocked by omega-conotoxin MVIIC (5 microM blocked approximately 70%). Bay K 8644 (10 microM) or omega-conotoxin GVIA (1 microM) had no significant effect on the alpha 1A current. Coexpression with rat brain Ca channel beta subunits increased the alpha 1A whole-cell current and shifted the current-voltage relation to more negative values. While the beta 1b and beta 3 subunits caused a significant acceleration of the alpha 1A inactivation kinetics, the beta 2a subunit dramatically slowed the inactivation of the alpha 1A current to that seen typically for P-type Ca currents. In situ localization with antisense deoxyoligonucleotide and RNA probes showed that alpha 1A was widely distributed throughout the rat central nervous system, with moderate to high levels in the olfactory bulb, in the cerebral cortex, and in the CA fields and dentate gyrus of the hippocampus. In the cerebellum, prominent alpha 1A expression was detected in Purkinje cells with some labeling also in granule cells. Overall, the results show that alpha 1A channels are widely expressed and share some properties with both Q- and P-type channels.

A beta-subunit normalizes the electrophysiological properties of a cloned N-type Ca2+ channel alpha 1-subunit.

The electrophysiological and pharmacological properties of a cloned rat brain N-type Ca2+ channel were determined by transient expression in Xenopus oocytes. Expression of the class B Ca2+ channel alpha 1 subunit, rbB-I, resulted in a high voltage-threshold current that activated slowly and showed little inactivation over 800 msec. Characteristic of N-type currents, the rbB-I current was completely blocked by omega-conotoxin GVIA and was insensitive to nifedipine and Bay K8644. The modulatory effects on the rbB-I current by cloned rat brain Ca2+ channel alpha 2 and beta 1b subunits were also examined. Coexpression of rbB-I with the beta 1b subunit caused significant changes in the properties of the rbB-I current making it more similar to N-type currents in neurons. These included: (1) an increase in the whole-cell current, (2) an increased rate of activation, (3) a shift of the voltage-dependence of inactivation to hyperpolarized potentials and (4) a pronounced inactivation of the current over 800 msec. Coexpression with the rat brain alpha 2 subunit had no significant effect on the rbB-I current alone but appeared to potentiate the rbB-I+beta 1b whole cell current. The results show that coexpression with the brain beta 1b subunit normalizes the rbB-I N-type current, and suggests the possibility that differences in subunit composition may contribute to the heterogeneous properties described for N-type channels in neurons.

Functional properties of a neuronal class C L-type calcium channel.

The rat brain class C calcium channel alpha 1 subunit cDNA, rbC-II, was subcloned into a vertebrate expression vector and transient expression was assayed following nuclear injection into Xenopus oocytes. Whole cell recordings showed that rbC-II currents (recorded with 40 mM Ba2+ as the charge carrier) had variable activation rates and minimal inactivation over an approximately 700 msec depolarizing step pulse. The pharmacological properties of the rbC-II current were consistent with those of an L-type calcium channel, being sensitive to dihydropyridines (10 microM nifedipine blocked approximately 85% of the current, 10 microM Bay K 8644 enhanced the current between 2- and 10-fold) and not affected by the N- and P-type calcium channel antagonists, omega-conotoxin GVIA and omega-agatoxin IVA, respectively. Coexpression of rbC-II with cloned rat neuronal calcium channel alpha 2 and beta subunits resulted in several changes to the electrophysiological properties of the rbC-II current including, an increased whole cell peak current, an increased rate of activation and a hyperpolarizing shift in the voltage dependence of activation. Taken together with results showing that the neuronal class D alpha 1 subunit also encodes an L-type calcium channel [Williams M. E., Feldman D. H., McCue A. F., Brenner R., Velicelebi G., Ellis S. B. and Harpold M. M. (1992a) Neuron 8: 71-84], these results indicate that the mammalian nervous system expresses two distinct genes encoding L-type calcium channels.

Structure and functional expression of a member of the low voltage-activated calcium channel family.

Oscillatory firing patterns are an intrinsic property of some neurons and have an important function in information processing. In some cells, low voltage-activated calcium channels have been proposed to underlie a depolarizing potential that regulates bursting. The sequence of a rat brain calcium channel alpha 1 subunit (rbE-II) was deduced. Although it is structurally related to high voltage-activated calcium channels, the rbE-II channel transiently activated at negative membrane potentials, required a strong hyperpolarization to deinactivate, and was highly sensitive to block by nickel. In situ hybridization showed that rbE-II messenger RNA is expressed in regions throughout the central nervous system. The electrophysiological properties of the rbE-II current are consistent with a type of low voltage-activated calcium channel that requires membrane hyperpolarization for maximal activity, which suggests that rbE-II may be involved in the modulation of firing patterns.

Hypoxia and N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, but not nerve growth factor, induce Na+ channels and hypertrophy in chromaffin-like arterial chemoreceptors.

Chronic hypoxia sensitizes the ventilatory reflex in mammals and causes enlargement of the carotid body, a peripheral arterial chemosensory organ. To investigate possible underlying mechanisms, in the absence of circulatory changes, we exposed cultures of dissociated rat carotid body containing the oxygen sensors (i.e., chromaffin-like glomus cells) to chronic hypoxia (6% O2) over a period of 2 weeks. After a delay of a few days, the Na+ current density in hypoxia-treated glomus cells increased significantly, reaching values up to 6 times that seen in normoxic (20% O2) controls. In addition the whole-cell capacitance, an indicator of cell size, was also significantly larger (3-4 times control) in glomus cells exposed to chronic hypoxia. Both effects were mimicked qualitatively by chronic treatment of normoxic cultures with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, but not nerve growth factor, which is known to induce similar changes in the chromaffin cell line PC12. Thus, the physiological and morphological effects of chronic hypoxia on the carotid body in vivo may be due in part to a cAMP-mediated stimulation of Na+ channel expression and hypertrophy in the chemosensory glomus cells.

Whole-cell currents in two subpopulations of cultured rat petrosal neurons with different tetrodotoxin sensitivities.

In this study we use whole-cell recording to characterize at least two distinct populations of cultured neurons from perinatal rat petrosal or petrosal/jugular ganglia based on differential sensitivity of the transient inward Na+ current to tetrodotoxin. These ganglia supply chemoreceptor and baroreceptor afferents which mediate several cardiovascular reflexes. Approximately 50% of the neurons sampled had Na+ currents that were virtually unaffected by bath addition of tetrodotoxin (0.5-2.0 microM) but were abolished by choline substitution for external Na+. The majority of the remaining neurons had Na+ currents that were rapidly and reversibly blocked by 500 nM tetrodotoxin. A few cells had both tetrodotoxin-resistant and tetrodotoxin-sensitive Na+ currents. All neurons had similar voltage-activated Ca2+ and K+ currents. The inward Ca2+ current had no obvious fast transient or T-type component and appeared to be due mainly to the presence of long-lasting L-type Ca2+ channels. The outward currents consisted largely of a delayed rectifying K+ current (IKdr) and a Ca(2+)-activated K+ current (IKca), but no obvious fast transient K+ current (IA) was observed. Exposure to a chemosensory stimulus, hypoxia (PO2 approximately 20 Torr), had no effect on these neurons, in contrast to the pronounced decrease in K+ current it produces in cultured glomus cells, the presumed chemoreceptors and normal targets for a subset of petrosal neurons in vivo. Current-clamp recordings indicated that some neurons gave single spikes while others gave multiple spikes in response to long-depolarizing stimuli. No correlation between spiking behaviour and tetrodotoxin-sensitivity was observed. Thus, cultures enriched in petrosal neurons contain subpopulations with differential sensitivities to tetrodotoxin. Since many of these neurons innervate a single chemosensory target organ, the carotid body, it is of interest to know whether one or both subtypes can form functional synapses with glomus cells of the carotid body and mediate a chemoreceptor reflex.

Effects of pHi and pHe on membrane currents recorded with the perforated-patch method from cultured chemoreceptors of the rat carotid body.

In this study we investigated the effects of intracellular pH (pHi) and extracellular pH (pHe) on whole-cell currents in cultured glomus cells of the rat carotid body and small, intensely fluorescent (SIF) cells of sympathetic ganglia. The use of the perforated-patch recording technique along with established methods of cytoplasmic acidification allowed us to carry out this study without greatly disturbing the cell's endogenous pH regulatory mechanisms. A reversible decrease in the outward K+ current (20-30%) was observed during acid loading of glomus (and SIF cells) using the K+/H+ ionophore nigericin (3 microM) and acetate (20 mM). A reversible decrease in the inward Na+ current was also observed in both cell types during nigericin application. Application of amiloride (0.1 mM) to the bathing solution inhibited recovery of the K+ current from an acid load implicating the Na+/H+ antiporter as a mechanism involved in pH homeostasis in glomus cells. A reversible decrease in K+ and Na+ currents was also observed during changes in pHe from 7.4 to 6.5. The effects of pHi on membrane currents, Ca2+ levels, and neurotransmitter release are discussed in the context of the role of glomus cells as primary transducers of chemosensory stimuli in arterial blood.

Contrasting effects of HEPES vs HCO3(-)-buffered media on whole-cell currents in cultured chemoreceptors of the rat carotid body.

In this study we compared the effects of physiological bicarbonate/CO2-buffered media (BBM) with the commonly used N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES)-buffered media (HBM) on whole-cell currents in cultured rat arterial chemoreceptors (i.e. glomus cells) using the perforated-patch technique. Two separate effects were observed on switching from HBM to BBM. First, in the majority of cells tested (31 of 36) there was an increase in the leakage conductance (ca. 5 fold) and a concomitant increase in channel noise, which in preliminary studies appears to arise from the opening of large-conductance anion channels. Second, there was a reversible decrease in voltage-activated outward K+ current which we attribute to cytoplasmic acidification, catalysed by carbonic anhydrase in glomus cells.

Whole-cell and perforated-patch recordings from O2-sensitive rat carotid body cells grown in short- and long-term culture.

We are investigating transduction mechanisms in a major peripheral chemosensory organ, the rat carotid body, using short- and long-term dissociated cell cultures and patch-clamp, whole-cell recording. In this study membrane properties of cultured glomus or type I cells were characterized with conventional whole-cell recording and the new perforated-patch technique during control (160 Torr) and low-PO2 (20 Torr) conditions. These cells contained voltage-gated channels typical of electrically excitable cells and had large input resistances (approx. 2 G omega). Under whole-cell voltage clamp the cells produced brief inactivating inward currents, which were largely abolished by 0.2-2.0 microM tetrodotoxin, followed by prolonged outward currents, which were reduced by 5 mM tetraethylammonium or abolished by the substitution of Cs+ ions for K+ ions in the pipette. On exposure to hypoxia the outward K+ current was reduced typically by 15%-20% with both conventional whole-cell and perforated-patch recording, which minimizes washout of the cell's cytoplasm. This effect persisted in long-term culture and was specific, since the inward current was unaffected and, moreover, it did not occur in cultured small intensely fluorescent cells, which are closely related to glomus cells. These properties of cultured rat glomus cells are contrasted with those recently reported for freshly isolated rabbit glomus cells.

Chloride channels in cultured glomus cells of the rat carotid body.

As part of our investigations on the chemosensory mechanisms in the rat carotid body, we are studying the physiology of the parenchymal glomus cells by the patch-clamp technique. Here we characterize a large-conductance chloride channel (approximately 296 pS) with random open and closed kinetics in inside-out patches of cultured glomus cells. The open-state probability (Po; mean = 0.61) was hardly affected by membrane potential (-50 to +50 mV) and cytoplasmic calcium (0-1 mM). Similarly, the channel did not appear to be regulated by cytoplasmic nucleotides (1 mM) or pH (6.5-8). Ion-substitution experiments yielded the following selectivity sequence: chloride greater than bicarbonate greater than sulfate greater than glutamate approximately sodium. Single-channel currents were reversibly reduced or blocked by anthracene-9-carboxylic acid (5-10 mM) but were unaffected by stilbene derivatives (0.5-1 mM), by furosemide (1 mM), and by 5-nitro-2-(3-phenyl-propylamino)benzoic acid (0.01 mM). Because these cultured glomus cells have been shown to express carbonic anhydrase, it is inferred that the chloride channels may play an important role in the physiology of glomus cells by aiding in the regulation of pHi and the resting potential via bicarbonate and chloride permeability.