H. D. Lux

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)

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.

Proton‐induced transformation of calcium channel in chick dorsal root ganglion cells.

1. In dissociated and cultured 2‐5‐day‐old chick dorsal root ganglion cells, a large transient inward current could be activated in response to a step increase in [H+]o. 2. Using the single‐electrode patch clamp technique in its whole‐cell configuration, the proton‐induced current was graded with [H+]o and relaxed in 1‐2 s. 3. The pH dependence of the current was sigmoid with activation occurring at around pH 7.0 (at[Ca2+]o = 1 mM) and a maximum at pH 6.0‐5.5. 4. Small increases of [H+]o, which by themselves failed to activate a significant amount of current, inactivated the proton‐induced current. The half‐maximum of inactivation occurred at pH 7.11 at [Ca2+]o = 5 mM, but this changed to pH 7.32 at [Ca2+]o = 1 mM. 5. The proton‐induced inward current reversed direction at the Na+ equilibrium potential and was suppressed in the absence of [Na+]o. Measurement of the reversal potential at different [Na+]o and/or [Na+]i showed a linear relation with a slope of 58 mV/decade as predicted from the Nernst equation. Thus, proton‐induced current was carried by Na+ and was abbreviated as INa(H). 6. The membrane conductance associated with INa(H) showed no voltage dependence, but did change in parallel with the activation of the current. The membrane conductance increased by a factor of 10‐20‐fold at the peak of the inward current. 7. INa(H) was blocked by organic and inorganic Ca2+ channel blockers (diltiazem, Cd2+ and Ni2+), but was unaffected by high concentrations of tetrodotoxin (TTX) or steady‐state increases of the [Ca2+]i to 10(‐4) M or the [H+]i to 10(‐6) M. 8. In outside‐out membrane patches, the single channel associated with the proton‐induced current opened in bursts, with long pauses. The mean open time during the bursts was 1.26 ms and the channel had a conductance of 20‐25 pS at ‐80 mV (120 mM [Na2+]o, 20 mM [Na2+]i). 9. Measurement of the voltage‐gated Ca2+ current using short (30‐50 ms) depolarizing pulses to zero showed that the Ca2+ current (ICa) but not the fast Na+ current (INa) was completely suppressed during the time course of activation of INa(H). 10. INa(H) was completely blocked by high (35‐40 mM) [Ca2+]o. 11. Simultaneous elevation of [H+]o and [Ca2+]o failed to activate INa(H) but enhanced the voltage‐gated Ca2+ channel. 12. Our data show that the proton‐induced current is carried by Na+ flowing through a transformed Ca2+ channel.(ABSTRACT TRUNCATED AT 400 WORDS)

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)

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.

Kinetics of GABAB receptor-mediated inhibition of calcium currents and excitatory synaptic transmission in hippocampal neurons in vitro

The time courses of the gamma-aminobutyric acid type B (GABAB) receptor-mediated inhibition of excitatory synaptic transmission and of action potential-evoked calcium currents were studied in hippocampal neurons in vitro with step-like changes of a saturating baclofen concentration. Inhibition mediated by postsynaptic GABAB receptors was excluded pharmacologically. Both presynaptic inhibition and reduction of calcium currents developed and declined exponentially with similar time constants of about 0.2 and 3 s, respectively. The close correlation of the time courses indicates that fast, G protein-mediated depression of voltage-gated calcium channels and thus direct reduction of the presynaptic calcium influx may contribute to the GABAB receptor-induced inhibition of excitatory synaptic transmission in hippocampal neurons in vitro.

Single channel activity associated with the calcium dependent outward current inHelix pomatia

A recently improved version of the extracellular patch clamp technique (9, 13) was used to record currents from microscopic membrane areas of Helix neurons with predominant Ca2+ dependent outward currents. Current fluctuations in the patches consisted mainly of frequently interrupted, one-sided steps indicating discrete open-closed state changes of single channels with an ohmic conductance of approximately 19 pS. Frequency of occurrence of the elementary events compares with amplitudes of macroscopic currents during depolarizing voltage steps of varied amplitude. Average delays in appearance of the events vary in line with delayed time courses of the cells outward current.

Voltage-dependent and calcium-dependent inactivation of calcium channel current in identified snail neurones.

1. The dependence of Ca2+ current inactivation on membrane potential and intracellular Ca2+ concentration ([Ca2+]i) was studied in TEA‐loaded, identified Helix neurones which possess a single population of high‐voltage‐activated Ca2+ channels. During prolonged depolarization, the Ca2+ current declined from its peak with two clearly distinct phases. The time course of its decay was readily fitted by a double‐exponential function. 2. In double‐pulse experiments, the relationship between the magnitude of the Ca2+ current and the amount of Ca2+ inactivation was not linear, and considerable inactivation was present, even when conditioning pulses were to levels of depolarization so great that Ca2+ currents were near zero. Similar results were obtained when external Ca2+ was replaced by Ba2+. 3. In double‐pulse experiments, hyperpolarization during the interpulse interval served to reprime a portion of the inactivated Ca2+ current for subsequent activation. The extent of repriming increased with hyperpolarization, reaching a maximum between ‐130 and ‐150 mV. The effectiveness of repriming hyperpolarizations was considerably increased when Ca2+ was replaced by Ba2+. 4. A significant fraction of inactivated Ca2+ channels can be recovered during hyperpolarizing pulses lasting only milliseconds. If hyperpolarizing pulses were applied before substantial inactivation of Ca2+ current, Ca2+ channels remained available for activation despite considerable Ca2+ entry. 5. The relationship between [Ca2+]i and inactivation was investigated by quantitatively injecting Ca2+‐buffered solutions into the cells. The time course of Ca2+ current inactivation was unchanged at free [Ca2+] between 1 x 10(‐7) and 1 x 10(‐5) M. From 1 x 10(‐7) to 1 x 10(‐9) M, inactivation became progressively slower, mainly due to a decrease of the amplitude ratio (fast/slow) of the two components of inactivation, which fell from about unity to near zero at 1 x 10(‐9) M. In double‐pulse experiments, recovery from inactivation was enhanced in neurones that had been injected with Ca2+ chelator. 6. We conclude that inactivation of Ca2+ channels in these neurones depends on both [Ca2+]i and membrane potential. The voltage‐dependent process may serve as a mechanism to quickly recover inactivated Ca2+ channels during repetitive firing despite considerable Ca2+ influx. 7. The results are discussed in the framework of a model which is based on two states of inactivation, INV and INCA, which represent different conformations of the inactivating substrate, and which are both reached from a lumped state of activation (A). Inactivation leads to high occupancy of INV during depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

The time courses of intracellular free calcium and related electrical effects after injection of CaCl2 into neurons of the snail, Helix pomatia.

Controlled quantities of 100 mM aqueous CaCl2 solutions were pressure injected into voltage-clamped neurons with a resolution of 10−11 1. Ca2+-selective microelectrodes monitored the time course of changes in [Ca2+]i. At a membrane potential of −50 mV CaCl2 quantities in the range of 1% of the cell volume induced an inward current, associated with a conductance increase and having an equilibrium potential between −20 and +20 mV, which accompanied the rise in [Ca2+]i. An artifactual origin of the inward current by the injection procedure or by calcium screening of membrane sites could be excluded. The calcium-induced hyperpolarizing conductance, producing an outward current at −50 mV, followed the inward current and reached maximum during the late decline in [Ca2+]i. In most cases its development was separated from the inward current by an intermediate relative decrease of the membrane conductance. Neither of the two transient conductance increases showed a particular dependence on voltage. Renewed Ca2+ injection quickly decreased the calcium-induced hyperpolarizing conductance for several seconds. Ca2+ injections below 0.05% of the cell volume mostly produced pure outward currents or hyperpolarizing responses. Partial substitution of extracellular CaCl2 by NiCl2 decreased the hyperpolarizing response but not the initial inward current. The immediate effects of increased [Ca2+]i are activation of a depolarizing conductance and the partial block of the late hyperpolarizing conductance. The latter is probably produced through intermediate steps after increasing [Ca2+i.

Site and mechanism of activation of proton‐induced sodium current in chick dorsal root ganglion neurones.

1. In dissociated and cultured 1‐ to 2‐day‐old chick dorsal root ganglion cells, and in isolated outside‐out membrane patches, a large transient current lasting 1‐2 s could be activated upon step increases in [H+]o. The proton‐induced current reversed direction at the Na+ equilibrium potential, was abolished completely in the absence of Na+, and was therefore labelled INa(H). 2. To investigate the activation and deactivation kinetics of INa(H) at the single‐channel level, we employed isolated membrane patches and a method whereby we could change the external solution in less than 1 ms. 3. In outside‐out membrane patches, INa(H) was fully activated within 2 ms between pH 6.7 and 5.7. Half‐times of activation decreased with increasing [H+]o. The calculated association rate constant was 9.5 x 10(9) M‐1 s‐1. 4. Deactivation of INa(H), following a step reduction in [H+]o, occurred with half‐times of within 1.3‐2 ms. 5. In the continued presence of an activating solution (pH 6.7 and 1 mM‐Ca2+), INa(H) inactivated slowly, with a time constant of about 300 ms. 6. Inactivation showed a limited dependence on [Ca2+]o. The time constant of inactivation increased from about 300‐500 ms as [Ca2+]o was decreased from 5 to 0.1 mM. Further decrease in [Ca2+]o did not significantly increase the time course of inactivation. Increases in [Ca2+]i from 10(‐9) to 10(‐3) M had no effect on the activation or inactivation kinetics of INa(H). 7. Conditioning proton concentrations which by themselves failed to activate any channel openings, partially inactivated INa(H). 8. Recovery from inactivation appeared to follow a time course similar to that of inactivation itself. 9. INa(H) could not be activated in inside‐out patches. A step increase in proton concentration outside a cell‐attached patch was also ineffective at producing INa(H) in the patch. Intracellular pH between 7.9 and 6.7 had no effect on the activation or inactivation of INa(H). 10. The activation and inactivation kinetics were not significantly voltage dependent. 11. The single‐channel conductance associated with the activation of INa(H) was 28 pS in symmetrical 120 mM‐NaCl solutions and remained constant throughout the time course of INa(H). 12. During activation of the voltage‐gated calcium current, ICa, a step increase in proton concentration caused a rapid (ca. 2 ms) suppression of ICa which was more than that predicted from the steady‐state effects of H+ on ICa. This effect was independent of [Na+] and the direction of INa(H).(ABSTRACT TRUNCATED AT 400 WORDS)