Anne Feltz

Depolarization elicits two distinct calcium currents in vertebrate sensory neurones.

The calcium currents of rat sensory neurones (of the IX and X cranial nerves) grown in culture were studied using whole cell recordings. In cells loaded with CsCl, and bathed in a solution where Na was replaced by choline or Tris, a step depolarization from −80 mV to 0 mV elicited the well-documented sustained Ca current (iCa,s). In contrast, depolarization from −80 mV to −60 mV and up to −20 mV evoked a distinct transient inward current (iCa,t) which could be isolated by imposing an internal pCa 7. It relaxed in about 100 ms and could possibly occur independently of the former current. The transient current was only affected by manipulation of the Ca concentration in the external medium and therefore was considered to be also a transfer of Ca. Ba was shown to act as a substitute with a lower affinity than Ca. The maximal amplitude of this current was in the order of a few hundred pA in Ca 5 mM and Mg 2 mM. Both activation and inactivation occurred in the same voltage span. The underlying event was studied using noise analysis and compared to the Ca transfer occurring during the sustained current as measured in chromaffin cells by Fenwick et al. (1982). We found them to be of similar amplitude.

Patch-clamp study of the tetrodotoxin-resistant sodium current in group C sensory neurones

Conditions were devised to isolate in cranial sensory neurones transfer of Na ions: K and Ca were omitted from the extracellular medium, and simultaneously cells were intracellularly loaded with 120 mM caesium and 20 mM TEA at [Ca]i = 10(-8) M. A tetrodotoxin (TTX)-resistant current was shown to be elicited by step depolarization from -25 MV upwards. This current successively activates and inactivates at increasing rates on further depolarization and at 0 mV (where peak amplitude is reached) its time course is of 20-50 ms. Absence of TTX-sensitivity (up to 15 microM), slow time course and an activation curve shifted by 15 mV towards the depolarized potentials differentiate this current from the more classical fast Na current which can be elicited on the same cells. Inactivation was provoked by a prepulse of varying amplitude and duration: with a prepulse command to -20 mV, inactivation was of 50% within a delay of 300 ms and almost 100% in about 1 min. After complete inactivation by command to 0 mV for 300 ms, recovery by holding the potential at -80 mV was of 50% in 205 ms, and of 100% after 1-4 s. It is concluded that a charge transfer of Na accounts for most of the hump which prolongs the action potential of these sensory neurones, and thus it can be proposed that spike duration as modulated by neurotransmitters may also involve Na in addition to Ca.

Molecular interaction of dihydropyridine receptors with type-1 ryanodine receptors in rat brain

In striated muscles, Ca2+ release from internal stores through ryanodine receptor (RyR) channels is triggered by functional coupling to voltage-activated Ca2+ channels known as dihydropyridine receptors (DHPRs) located in the plasma membrane. In skeletal muscle, this occurs by a direct conformational link between the tissue-specific DHPR (Ca(v)1.1) and RyR(1), whereas in the heart the signal is carried from the cardiac-type DHPR (Ca(v)1.2) to RyR(2) by calcium ions acting as an activator. Subtypes of both channels are expressed in the central nervous system, but their functions and mechanisms of coupling are still poorly understood. We show here that complexes immunoprecipitated from solubilized rat brain membranes with antibodies against DHPR of the Ca(v)1.2 or Ca(v)1.3 subtypes contain RyR. Only type-1 RyR is co-precipitated, although the major brain isoform is RyR(2). This suggests that, in neurons, DHPRs could communicate with RyRs by way of a strong molecular interaction and, more generally, that the physical link between DHPR and RyR shown to exist in skeletal muscle can be extended to other tissues.

Distinct kinetics of cloned T-type Ca2 + channels lead to differential Ca2 + entry and frequency-dependence during mock action potentials.

Voltage‐dependent activity around the resting potential is determinant in neuronal physiology and participates in the definition of the firing pattern. Low‐voltage‐activated T‐type Ca2 +  channels directly affect the membrane potential and control a number of secondary Ca2 + ‐dependent permeabilities. We have studied the ability of the cloned T‐type channels (α1G,H,I) to carry Ca2 +  currents in response to mock action potentials. The relationship between the spike duration and the current amplitude is specific for each of the T‐type channels, reflecting their individual kinetic properties. Typically the charge transfer increases with spike broadening, but the total Ca2 +  entry saturates at different spike durations according to the channel type: 4 ms for α1G; 7 ms for α1H; and >  10 ms for α1I channels. During bursts, currents are inhibited and/or transiently potentiated according to the α1 channel type, with larger effects at higher frequency. The inhibition may be induced by voltage‐independent transitions toward inactivated states and/or channel inactivation through intermediate closed states. The potentiation is explained by an acceleration in the channel activation kinetics. Relatively fast inactivation and slow recovery limit the ability of α1G and α1H channels to respond to high frequency stimulation ( >  20 Hz). In contrast, the slow inactivation of α1I subunits allows these channels to continue participating in high frequency bursts (100 Hz). The biophysical properties of α1G, H and I channels will therefore dramatically modulate the effect of neuronal activities on Ca2 +  signalling.

Voltage-dependent transient calcium currents in freshly dissociated capillary endothelial cells

Dissociated capillary endothelial cells display a voltage‐dependent Ca current activating around the resting potential. The initial transient component of the current corresponds to a Ca channel of the T type. Some cells also display a plateau component corresponding to a distinct dihydropyridine‐sensitive Ca channel. Depolarization induced by high external K+ elicits an increase in cytoplasmic Ca concentration. Confluent cells have been found to express the same Ca permeabilities.

Spontaneous and GABA-evoked chloride channels on pituitary intermediate lobe cells and their internal Ca requirements.

On porcine intermediate lobe (IL) endocrine cells, spontaneously opening chloride channels have been studied and compared to GABA-A activated chloride channels. Elementary currents were recorded mainly from outside-out patches excised from IL cells maintained in culture for 1–4 weeks. Spontaneous inward currents were observed in Cs-loaded cells after replacing Na in the extracellular medium by the impermeant ion choline. This activity, at an internal calcium concentration of 10−8 M corresponded to a channel for chloride ions with a main conductance level of 26 pS, and substates around 11 pS. The sequence of permeabilities to halides was I>Br>Cl. These conductance characteristics were common to the GABA-operated channels which also showed a main conductance substate of 23–31 pS. The open time of the 26 pS level mostly encountered in spontaneous activity, was distributed along two modes: one, the most frequent, around 1 ms, and the other around 4 ms. This latter mode was the predominant one observed during GABA and isoguvacine applications but in addition a bursting activity of 19 ms duration was also seen. Specific GABA-A receptor antagonists (bicuculline and SR 42641, 1 μM) blocked activity evoked by GABA (1–10 μM), but did not affect spontaneous events. These spontaneous Cl events were only observed in a restricted range of internal Ca concentrations, i.e. between 1 nM and 0.1 μM, and were practically abolished at Cai 1 μM. The GABA-induced activity of Cl channels was also Ca-sensitive, being reduced when Cai reached 1 μM.

An analysis of acetylcholine responses of junctional and extrajunctional receptors of frog muscle fibres. With an Appendix

1. An analysis has been made of the distribution and rise time of the responses induced by electrophoretic application of ACh on junctional and extrajunctional receptors of the neuromuscular junction of the frog.

Voltage-gated Ca entry in isolated bovine capillary endothelial cells: evidence of a new type of BAY K 8644-sensitive channel

Isolated bovine capillary endothelial cells have been examined for voltage-dependent Ca entry. All cells displayed a low threshold activity, with the main characteristics of a T-type transient current, when examined using whole-cell recording for activation and inactivation and cell-attached conditions or inside-out patches for the elementary conductance (8 pS). 25% of the cells displayed an additional sustained current in 5 mM CaCl2 above −40 mV, which was enhanced by application of BAY K 8644, but almost insensitive to superfusion with nicardipine. Two types of channels (2.8 and 21 pS, in 110 mM BaCl2) were shown to have a BAY K 8644 sensitivity. The large conductance channels were L-type channels. The smaller events were elicited at more hyperpolarized potentials (by some 30 mV). Their mean open time was 16 ms in control conditions. In presence of BAY K 8644, additional long open times were observed (up to 100 ms as compared to 7.8 ms for the time constants of the slow mode of the L-type channel). We refer to these channels as SB channels: of small conductance and sensitive to BAY K 8644. In the presence of nicardipine, SB channels are not noticeably modified, in contrast to the L-type openings which are abolished. Also, SB open times are close to control values when nicardipine is added after a BAY K 8644 application. We suggest that, at physiological concentrations of divalent ions, an SB-type activity is elicited above −40 mV which generates the low threshold sustained current.

Two types of calcium channels are expressed in adult bovine chromaffin cells

1. Calcium channel activity was recorded in chromaffin cells in the cell‐attached condition, using 110 mM‐Ba2+ as the permeant ion. 2. One type of calcium channel had a conductance of 16 pS, was completely inactivated at a holding potential of ‐20 mV and was insensitive to dihydropyridine agonists and antagonists. These characteristics correspond to a calcium channel of the N‐type. 3. A second type of calcium channel was active at holding potentials of ‐30 mV and above, had a channel conductance of 31 pS, and was sensitive to the dihydropyridine agonist, Bay K 8644. The channel opened along two dominant modes with characteristic time constants of 0.5 and 5 ms. The main effect of Bay K 8644 was to increase the probability of both short and long openings with no change in their relative proportions (6 to 1 respectively). These characteristics correspond to a calcium channel of the L‐type. 4. omega‐Conotoxin affected the activity of both N‐ and L‐type channels. It drastically reduced the number of N‐type channel openings and produced complex changes in L‐type channel activity. Long openings were less frequent and the conductance during short openings was slightly smaller than that measured in the presence of Bay K 8644. 5. The discussion focuses on modulation of L‐type channel activity. Openings of L‐type channels are rarely recorded in the cell‐attached configuration under control conditions. Addition of Bay K 8644 is needed to reveal the presence of L‐type channels. By contrast, L‐type currents recorded in the whole‐cell configuration are always observed and are insensitive to Bay K 8644. These results indicate that L‐type channels are normally inoperable in chromaffin cells.

Polyethylenimine-Mediated DNA Transfection of Peripheral and Central Neurons in Primary Culture: Probing Ca2+Channel Structure and Function with Antisense Oligonucleotides

To study neuronal ion channel function with antisense oligonucleotides, a reliable method is needed which allows different neuronal cell types to be transfected without artifactual disruptive effects on their electrical properties. Here we report that use of the recently introduced transfecting agent, polyethylenimine, fulfills this requirement. Four days after transfection, in both central and peripheral neurons, an antisense designed to block the synthesis of the Ca2+ channel beta subunits induced a maximal decrease of the Ca2- current amplitude and modification of their kinetics and voltage-dependence. Controls with scrambled oligonucleotides, as well as Na+ current recordings of antisense transfected neurons, confirm both that the transfecting agent does not modify the electrophysiological properties of the neurons and that the effect of the antisense is sequence specific.