Emilio Carbone

Kinetics and selectivity of a low-voltage-activated calcium current in chick and rat sensory neurones.

1. Using the whole‐cell recording mode of the patch‐clamp technique, we have investigated kinetic and selectivity properties of a low‐voltage‐activated (l.v.a.) Ca2+ current in chick and rat dorsal root ganglion (d.r.g.) neurones. 2. L.v.a currents were activated at about ‐50 mV and reached maximum amplitudes between ‐30 and ‐20 mV with averages of ‐0.16 nA in chick and ‐0.3 nA in rat d.r.g. cells with 5 mM‐extracellular Ca2+. Between ‐60 and ‐20 mV, the time to peak, tp, of this current decreased with increasing membrane depolarizations. An e‐fold change of tp required a 14 mV potential change in chick and a 17 mV change in rat d.r.g. cells at 22 degrees C. 3. Between ‐50 and +20 mV inactivation of this current was fast, single exponential and voltage dependent. In rat, the time constant of inactivation, tau h, was smaller and less voltage dependent than in chick. 4. The amplitude of these currents increased by a factor of 5‐10, when the extracellular Ca2+ concentration was changed from 1 to 95 mM. Amplitudes and kinetic parameters of the currents showed typical shifts along the voltage axis. No correlation between Ca2+ current amplitudes and activation‐inactivation kinetics was found, suggesting that the reaction rates which control these processes are not dependent on Ca2+ entry. 5. Recovery from inactivation was voltage dependent and developed with a time constant, tau r, in the order of 1 s. tau r was nearly halved by changing the potential from ‐80 to ‐120 mV. 6. Tail currents associated with membrane repolarization were also voltage dependent and developed exponentially. Their time constant decreased by a factor of 3 when the potential was changed from ‐60 to ‐100 mV. 7. A second and more prominent Ca2+ current was activated at potentials positive to ‐20 mV (high‐voltage‐activated Ca2+ currents, h.v.a.), masking the time course of l.v.a. currents. Between ‐20 and 0 mV, time to peak of the entire current increased by a factor of 2 but decreased again at higher membrane potentials. Inactivation also became significantly slower in this potential range. 8. The contribution of the h.v.a. component to the total membrane current was markedly reduced using a high intracellular Ca2+ concentration, [Ca2+]i, or internal fluoride salts. This made it possible to study the kinetic parameters and the I‐V characteristics of the l.v.a. current more precisely over a wider potential range (‐50 to +30 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of dopamine and noradrenaline on Ca channels of cultured sensory and sympathetic neurons of chick

The effects of noradrenaline and dopamine on voltage-dependent Ca currents were investigated in cultured dorsal root and sympathetic ganglion neurons from chick embryos. At concentrations of 1 to 10 μM, bath application of the neurotransmitters caused a general depression of inward Ca currents. Above −20 mV the decrease of the current amplitude was reversible and accompanied by a 2–10-fold prolongation of the activation time course. Below −20 mV, where a low voltage-activated Ca component is turned on, the size of the currents was reduced by 40% with little effect on the time course. Despite extensive wash-out, little sign of reversibility was observed in this case.Single-channel current recording in outside-out membrane patches revealed that a low membrane potentials dopamine and noradrenaline reversibly reduced single Ca-channel activity. This finding supports the view that in sensory and sympathetic neurons, both neurotransmitters affect the membrane conductance by modulating Ca permeability and not by activating catecholamine-specific channels able to carry transient outward currents. The probability of Ca channel opening is strongly reduced by addition of 10 μM of either catecholamine to the bath. The possible involvement of a voltage-dependent block of Ca channels by the neurotransmitters is discussed.

A low voltage-activated calcium conductance in embryonic chick sensory neurons

Isolated Ca currents in cultured dorsal root ganglion (DRG) cells were studied using the patch clamp technique. The currents persisted in the presence of 30 microM tetrodotoxin (TTX) or when external Na was replaced by choline. They were fully blocked by millimolar additions of Cd2+ and Ni2+ to the bath. Two components of an inward-going Ca current were observed. In 5 mM external Ca, a current of small amplitude, turned on already during steps changes to -60 mV membrane potential, leveled off at -30 mV to a value of approximately 0.2 nA. A second, larger current component, which resembled the previously described Ca current in other cells, appeared at more positive voltages (-20 to -10 mV) and had a maximum approximately 0 mV. The current component activated at the more negative membrane potentials showed the stronger dependence on external Ca. The presence of a time- and a voltage-dependent activation was indicated by the currents sigmoidal rise, which became faster with increased depolarization. Its tail currents were generally slower than those associated with the Ca currents of larger amplitude. From -60 mV holding potential, the maximum obtainable amplitude of the low depolarization-activated current was only one-tenth of that achieved from a holding potential of -90 mV. Voltage-dependent inactivation of this current component was fast compared with that of the other component. The properties of this low voltage-activated and fully inactivating Ca current suggest it is the same as the inward current that has been postulated in several central neurons (Llinas, R., and Y. Yarom, 1981, J. Physiol. (Lond.), 315:569-584), which produce depolarizing potential waves and burst-firing only when membrane hyperpolarization precedes.

Do calcium channel classifications account for neuronal calcium channel diversity

Calcium (Ca2+) ions are involved in the development and control of a variety of neuronal properties and functions such as channel expression, synaptic transmission and neurosecretion. The main pathway by which Ca2+ enters the intracellular space is through voltage-activated Ca2+ channels that can be classified according to their different biophysical and pharmacological properties. Identification and characterization of these channel types are prerequisites for understanding the mechanisms that underlie Ca2(+)-controlled processes. In this article we summarize the efforts made to identify neuronal Ca2+ channel types, and we attempt to evaluate how useful existing classifications are in assigning specific properties and functions to distinct channel types in neurons.

Single low-voltage-activated calcium channels in chick and rat sensory neurones.

1. Single and multiple Ca2+ channel currents were recorded from outside‐out and cell‐attached patches of cultured chick and rat dorsal root ganglion cells, using the patch‐clamp technique. 2. Outside‐out patches containing a large number of Ca2+ channels revealed Ca2+ currents resembling those from the whole cell. A low‐voltage‐activated (l.v.a.) and a high‐voltage‐activated (h.v.a.) Ca2+ current similar to those described in the accompanying paper (Carbone & Lux, 1987 b) could be distinguished. The h.v.a. current component subsided within 10 min following the formation of the patch, while the l.v.a. component lasted much longer. 3. Unitary events related to the l.v.a. Ca2+ channel could be clearly resolved in outside‐out patches formed in Na+‐ and K+‐free media containing 5‐50 mM‐CaCl2. 4. The amplitudes of l.v.a. channel openings were bimodally distributed, indicating the presence of two conductive states. At ‐40 mV, mean amplitudes of the two events were ‐0.29 +/‐ 0.07 pA and ‐0.47 +/‐ 0.085 pA in 50 mM‐CaCl2, with apparent slope conductances of about 3.6 and 5.2 pS, respectively. In 5 mM‐CaCl2 both slope conductances were about 3 times smaller. The mean open times were similar for both states and were fitted by a simple exponential with a time constant of about 2.5 ms at ‐40 mV. The time constant decreased with more‐negative membrane potentials and was 0.9 ms at ‐100 mV. Openings frequently occurred in bursts separated by longer‐lasting closures. The mean closed time during bursts was 1.33 ms at ‐40 mV. 5. Time and amplitude distributions of elementary events were similar for chick and rat sensory neurones and with Ba2+ and Sr2+ replacing external Ca2+. 6. In the potential range examined (from ‐60 to ‐30 mV), the first‐latency distribution function revealed a distinct rise to peak which occurred at considerably earlier times than peaks of macroscopic currents. The time course of macroscopic l.v.a. Ca2+ currents could be simulated in two ways: (a) by using a five‐state Markov‐chain model with rate constants estimated from the transition probabilities and dwell times of the channel states, and (b) by evaluating the convolution integral of the first‐latency function and the burst open probability of the channel. Both approaches suggest that activation and inactivation are weakly coupled and that the l.v.a. channel of sensory neurones reopens several times before inactivating.(ABSTRACT TRUNCATED AT 400 WORDS)

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 lowthreshold (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 μM ω-conotoxin (ω-CgTx) or 40 μM 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-ω-CgTx binding sites increased markedly during cell differentiation. In agreement with recent reports, 6.4 μM ω-CgTx blocked only about 85% of the Ba currents through HVA channels in 50% of the cells. Residual ω-CgTx-resistant currents proved to be more sensitive to dihydropyridines (DHP) than total HVA currents. Bay K 8644 (1 μM) had a clear agonistic action on ω-CgTx-resistant currents and was preferred to other Ca antagonists for identifying HVA DHP-sensitive channels. Compared to the ω-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.

Loss of Cav1.3 Channels Reveals the Critical Role of L-Type and BK Channel Coupling in Pacemaking Mouse Adrenal Chromaffin Cells

We studied wild-type (WT) and Cav1.3−/− mouse chromaffin cells (MCCs) with the aim to determine the isoform of L-type Ca2+ channel (LTCC) and BK channels that underlie the pacemaker current controlling spontaneous firing. Most WT-MCCs (80%) were spontaneously active (1.5 Hz) and highly sensitive to nifedipine and BayK-8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid, methyl ester). Nifedipine blocked the firing, whereas BayK-8644 increased threefold the firing rate. The two dihydropyridines and the BK channel blocker paxilline altered the shape of action potentials (APs), suggesting close coupling of LTCCs to BK channels. WT-MCCs expressed equal fractions of functionally active Cav1.2 and Cav1.3 channels. Cav1.3 channel deficiency decreased the number of normally firing MCCs (30%; 2.0 Hz), suggesting a critical role of these channels on firing, which derived from their slow inactivation rate, sizeable activation at subthreshold potentials, and close coupling to fast inactivating BK channels as determined by using EGTA and BAPTA Ca2+ buffering. By means of the action potential clamp, in TTX-treated WT-MCCs, we found that the interpulse pacemaker current was always net inward and dominated by LTCCs. Fast inactivating and non-inactivating BK currents sustained mainly the afterhyperpolarization of the short APs (2–3 ms) and only partially the pacemaker current during the long interspike (300–500 ms). Deletion of Cav1.3 channels reduced drastically the inward Ca2+ current and the corresponding Ca2+-activated BK current during spikes. Our data highlight the role of Cav1.3, and to a minor degree of Cav1.2, as subthreshold pacemaker channels in MCCs and open new interesting features about their role in the control of firing and catecholamine secretion at rest and during sustained stimulations matching acute stress.

Effect of menthol on two types of Ca currents in cultured sensory neurons of vertebrates

The effect of menthol on voltage-dependent Ca currents was investigated in cultured dorsal root ganglion cells from chick and rat embryos. Bath application of menthol (0.1–1 mM) had different effects on the various Ca currents present in these neurons. Below −20 mV, the low threshold Ca currents were reduced in amplitude in a dose-dependent manner by menthol with little changes of their activation kinetics. In contrast to this, the time course of inactivation of the high-threshold Ca currents, activated above −20 mV from a holding potential of −80 mV, was drastically accelerated by external menthol. The action of menthol was unchanged with more positive holding potentials (−50 mV). Thus, a proposed third type of Ca current with transient activation and complete deactivation below −50 mV was either not present or not affected by menthol. Menthol exerted its action only when applied from the outside. Its effect was completely reversible within 15–20 min of wash-out. Our findings are consistent with the idea that menthol acts on two types of Ca channels coexisting on the membrane of cultured sensory neurons. Menthol blocks currents through the low voltage-activated Ca channel, and facilitates inactivation gating of the classical high voltage-activated Ca channel.

Chronic hypoxia up‐regulates α1H T‐type channels and low‐threshold catecholamine secretion in rat chromaffin cells

α1H T‐type channels recruited by β1‐adrenergic stimulation in rat chromaffin cells (RCCs) are coupled to fast exocytosis with the same Ca2+ dependence of high‐threshold Ca2+ channels. Here we show that RCCs exposed to chronic hypoxia (CH) for 12–18 h in 3% O2 express comparable densities of functional T‐type channels that depolarize the resting cells and contribute to low‐voltage exocytosis. Following chronic hypoxia, most RCCs exhibited T‐type Ca2+ channels already available at −50 mV with the same gating, pharmacological and molecular features as the α1H isoform. Chronic hypoxia had no effects on cell size and high‐threshold Ca2+ current density and was mimicked by overnight incubation with the iron‐chelating agent desferrioxamine (DFX), suggesting the involvement of hypoxia‐inducible factors (HIFs). T‐type channel recruitment occurred independently of PKA activation and the presence of extracellular Ca2+. Hypoxia‐recruited T‐type channels were partially open at rest (T‐type ‘window‐current’) and contributed to raising the resting potential to more positive values. Their block by 50 μm Ni2+ caused a 5–8 mV hyperpolarization. The secretory response associated with T‐type channels could be detected following mild cell depolarizations, either by capacitance increases induced by step depolarizations or by amperometric current spikes induced by increased [KCl]. In the latter case, exocytotic bursts could be evoked even with 2–4 mm KCl and spike frequency was drastically reduced by 50 μm Ni2+. Chronic hypoxia did not alter the shape of spikes, suggesting that hypoxia‐recruited T‐type channels increase the number of secreted vesicles at low voltages, without altering the mechanism of catecholamine release and the quantal content of released molecules.

K+ conductance modified by a titratable group accessible to protons from the intracellular side of the squid axon membrane

In the range of pH examined (5.2-10), variations of internal pH from high to low values result in a reversible decrease of the conductance of the open K channels, without significantly affecting the kinetics parameters. A linear plot of the conductance versus internal pH suggests the existence of a titratable group that has an apparent pKa of about 6.9, and that is accessible to protons only from the intracellular side of the membrane.