Roland Bournaud

Contryphan-Vn: a modulator of Ca2+-dependent K+ channels ☆

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.

Low threshold T‐type calcium current in rat embryonic chromaffin cells

1 The gating kinetics and functions of low threshold T‐type current in cultured chromaffin cells from rats of 19–20 days gestation (E19‐E20) were studied using the patch clamp technique. Exocytosis induced by calcium currents was monitored by the measurement of membrane capacitance and amperometry with a carbon fibre sensor. 2 In cells cultured for 1–4 days, the embryonic chromaffin cells were immunohistochemically identified by using polyclonal antibodies against dopamine β‐hydroxylase (DBH) and syntaxin. The immuno‐positive cells could be separated into three types, based on the recorded calcium current properties. Type I cells showed exclusively large low threshold T‐type current, Type II cells showed only high voltage activated (HVA) calcium channel current and Type III cells showed both T‐type and HVA currents. These cells represented 44 %, 46 % and 10 % of the total, respectively. 3 T‐type current recorded in Type I cells became detectable at −50 mV, reached its maximum amplitude of 6.8 ± 1.2 pA pF−1 (n= 5) at −10 mV and reversed around +50 mV. The current was characterized by criss‐crossing kinetics within the −50 to −30 mV voltage range and a slow deactivation (deactivation time constant, τd= 2 ms at −80 mV). The channel closing and inactivation process included both voltage‐dependent and voltage‐independent steps. The antihypertensive drug mibefradil (200 nm) reduced the current amplitude to about 65 % of control values. Ni2+ also blocked the current in a dose‐dependent manner with an IC50 of 25 μm. 4 T‐type current in Type I cells did not induce exocytosis, while catecholamine secretion by exocytosis could be induced by HVA calcium current in both Type II and Type III cells. The failure to induce exocytosis by T‐type current in Type I cells was not due to insufficient Ca2+ influx through the T‐type calcium channel. 5 We suggest that T‐type current is expressed in developing immature chromaffin cells. The T‐type current is replaced progressively by HVA calcium current during pre‐ and post‐natal development accompanying the functional maturation of the exocytosis mechanism.

Catecholamine secretion from rat foetal adrenal chromaffin cells and hypoxia sensitivity

The adrenal medulla chromaffin cells (AMCs) secrete catecholamines in response to various types of stress. We examined the hypoxia-sensitivity of catecholamine secretion by rat foetal chromaffin cells in which the innervation by the splanchnic nerve is not established. The experiments were performed in primary cultured cells from two different ages of foetuses (F15 and F19). Membrane potential of AMCs was monitored with the patch clamp technique, and the catecholamine secretion was detected by amperometry. We found that: (1) AMCs from F19 foetuses showed hypoxia-induced catecholamine release. (2) This hypoxia-induced secretion is produced by membrane depolarization generated by an inhibition of Ca2+-activated K+ current [IK(Ca)] current. (3) Chromaffin precursor cells from F15 foetuses secrete catecholamine. The quantal release is calcium-dependent, but the size of the quantum is reduced. (4) In the precursor cells, a hypoxia-induced membrane hyperpolarization is originated by an ATP-sensitive K+ current [IK(ATP)] activation. (5) During the prenatal period, at F15, the percentage of the total outward current for IK(ATP) and IK(Ca) was 50 and 29.5%, respectively, whereas at F19, IK(ATP) is reduced to 14%, and IK(Ca) became 64% of the total current. We conclude that before birth, the age-dependent hypoxia response of chromaffin cells is modulated by the functional activity of KATP and KCa channels.

Development of multiple calcium channel types in cultured mouse hippocampal neurons

The development of multiple calcium channel activities was studied in mouse hippocampal neurons in culture, using the patch-clamp technique. A depolarizing pulse (40-50 ms duration) from the holding potential of -80 mV to levels more depolarized than -40 mV produced a low threshold T-type current. The T-type current was observed in 52% of four days in vitro neurons. The number of neurons which expressed T-type current decreased with age of culture, so that the current was detected in only 18% of neurons after 16 days in vitro. The T-type current densities varied between 1.9 pA/pF and 3.29 pA/pF in the mean values during the period studied (4-16 days in vitro). A depolarizing pulse from -80 mV to levels more depolarized than -35 mV evoked a high threshold calcium channel current. The high threshold current density increased in the mean values from 3.9 pA/pF in four days in vitro neurons to 28 pA/pF in 16 days in vitro neurons. We have then examined the effect of nifedipine, omega-Agatoxin IVA and omega-conotoxin GVIA on the high threshold current. Nifedipine (1-5 microM) sensitive current density stayed in the range of 1.9-2.1 pA/pF during 4-16 days in vitro, while omega-Agatoxin IVA (200 nM) sensitive current density increased in the mean values from 1.54 pA/pF in four days in vitro neurons to 21.5 pA/pF in 16 days in vitro neurons. The omega-conotoxin GVIA sensitive N-type channel current was maximum at eight days in vitro (5.44 pA/pF) and it reduced progressively to reach almost half (2.46 pA/pF) in 16 days in vitro neurons. These results showed that diverse subtypes of calcium channels change in density during the early period of culture. We suggest that the temporal expression of each type of channel may be linked to the development of neural activities.

Appearance of the slow Ca conductance in myotubes from mutant mice with “muscular dysgenesis”

Voltage gated Ca conductance in skeletal muscle cells from mice with muscular dysgenesis (mdg/mdg) and from normal mice was studied using the whole cell recording technique. The physiological properties of the myotubes from the mutant mice (uncoupling of excitation-contraction, deficiency in the voltage gated slow Ca conductance) were changed to normal when themdg/mdg myotubes were cocultured with spinal cord cells from normal mice. Spinal cord cells from mutant mice failed to induce normal muscle activity in the mutant myotubes. In aged mutant myotubes cultured without spinal cord cells, the slow Ca conductance sometimes developed, although with smaller amplitude. The number ofmdg/mdg myotubes with partial development of the slow Ca conductance increased with the age of the culture. E-C coupling was never established in aged mutant myotubes. The phenotypic reversion did not require functional synaptic transmission since it was also obtained when neuromuscular transmission was chronically blocked with α-bungarotoxin (4–40 μg/ml).

Intramembrane charge movement in developing skeletal muscle cells from fetal mice

The development of intramembrane charge movement was studied in freshly isolated skeletal muscle cells from 13- to 19-day-old mouse fetuses. Charge movement was present in myotubes from 13-day-old fetuses. The relationship between charge movement and membrane potential could be described by a two-state Boltzmann equation. The amount of maximum charge movement (Qmax) increased substantially with the age of the fetuses from 2.84±0.39 nC/μF (n=10) at day 13 to 10.01±0.97 nC/μF (n=15) at day 19. Nifedipine (1 μM) consistently reduced Qmax by 33±2% (n=37) of the control value at each age studied. Increasing the concentration of nifedipine to 20 μM had no further effect, suggesting that the charge movement in developing myotubes consists of at least two components: a nifedipine-sensitive charge movement (Qns) and a nifedipine-resistant one (Qnr). Both Qns and Qnr increased exponentially with a distinct enhancement of rate at day 16.

Nifedipine-sensitive intramembrane charge movement in Purkinje cells from mouse cerebellum.

1. The intramembrane charge movement was recorded in freshly dissociated Purkinje cells from 14‐ to 18‐day‐old mouse cerebellum using the whole‐cell voltage clamp technique. 2. After pharmacological elimination of all ionic currents, a depolarizing pulse from a holding potential of ‐80 mV revealed a transient capacitive outward current at the onset and a transient inward current at the end of the pulse. The amount of charge transferred at the onset (Qon) was equivalent to that moved at the end of the pulse (Qoff). The decay time course of Qon can be fitted by a single exponential curve with a maximum time constant of 1.89 +/‐ 0.35 ms at 20 mV (n = 11). 3. The charge movement had an S‐shaped dependence on test membrane potential, according to a two‐state Boltzmann function. The maximum amount (Qmax) of Qon that could be moved was 17.46 +/‐ 0.83 nC muF‐1; the membrane potential at which half the charge movement occurred (V) was 13.48 +/‐ 2.20 mV and the slope factor (k) was 16.83 +/‐ 0.84 mV (n = 27). 4. Phenylglyoxal (2 mM), an arginine‐specific modifying reagent, reduced Qmax to 60% of control after 20 min treatment. 5. The charge movement was partially immobilized by nifedipine in a dose‐dependent manner with an IC50 of 70 nM. The fraction of the nifedipine‐sensitive component was 39% of the total charge movement. The potential dependence of the nifedipine‐sensitive charge movement could be expressed by a Boltzmann function with values of 7.00 +/‐ 0.53 nC muF‐1 for Qmax, 31.44 +/‐ 4.23 mV for V and 21.53 +/‐ 3.18 mV for k (n = 8). 6. The P‐type calcium channel specific inhibitor, omega‐Aga IVA (250 nM), had no effect on intramembrane charge movement. 7. The above results show that part of the intramembrane charge movement in Purkinje cells may be related to a conformational change of DHP receptors upon membrane depolarization.

Reduced intramembrane charge movement in the dysgenic skeletal muscle cell.

Intramembrane charge movement in skeletal muscle cells has been proposed to underlie the process leading to Ca release from the sarcoplasmic reticulum. A number of recent studies suggest that the dihydropyridine receptor located in the transverse-tubular membrane is responsible for the generation of intramembrane charge movement. The skeletal muscle cell of the mutant mouse with “Muscular Dysgenesis” is characterized by absence of excitation-contraction coupling. Here we investigated the charge movement in freshly dissociated skeletal muscle cells from dysgenic mice. In 9 out of 34 dysgenic mouse cells the charge movement was completely absent, in the remaining cells the charge movement was never more than 30 % of control. The amount of maximum charge movement (Qmax) in mutant muscle cells was less than 30 % of Qmax in normal muscle. Nifedipine, a dihydropyridine derivative, reduced the amount of charge movement in normal muscle cells but it was less effective on charge movement in mutant muscle cells. We conclude that there is an alteration of nifedipine-sensitive charge movement in the skeletal muscle cells from the mutant mice.

Extracellular Ca2+-dependent and independent calcium transient in fetal myotubes

Spatio-temporal changes in the intracellular calcium concentration [Ca2+]i of dissociated mice myotubes from 14-day and 18-day-old fetuses were studied using digital imaging analysis of the Ca2+ indicator fura-2. Myotubes from 18-day-old fetuses displayed a transient [Ca2+]i increase upon electrical stimulation either in nominally calcium-free external solution or in Krebs solution containing 100 μM lanthanum. Thus, at this developmental stage, membrane depolarization appears to increase [Ca2+]i by stimulating Ca2+ release from the sarcoplasmic reticulum independently of extracellular Ca2+ influx. Similarly, myotubes from 14-day-old fetuses also showed a calcium transient upon electrical stimulation in Krebs solution. However, in 46% of these myotubes the calcium transient was abolished when Ca2+ entry through calcium channels was suppressed.

Asymmetric intramembrane charge movement in mouse hippocampal pyramidal cells

Intramembrane charge movement was recorded from freshly dissociated hippocampal pyramidal cells from mice using the whole cell clamp technique. Once the ionic currents were suppressed, a depolarizing pulse from a holding potential of -80 mV elicited a capacitive transient outward current at onset and a capacitive inward current at offset of the pulse. The amount of charge displaced at the onset of the pulse (Qon) was equivalent to the charge moved at repolarization (Qoff). The relationship between the amount of charge moved and pulse potential could be expressed by a simple two states Boltzmann equation: Q = Qmax/(1 + exp[-(V-V1/2)/k]), where Qmax is the maximum charge, V1/2 the membrane potential at which Q is half of Qmax and k is a slope factor. On average, Qmax was 10.90 +/- 0.62 nC/microF, V1/2 was 1.70 +/- 2.90 mV, and k was 18.80 +/- 1.20 mV (n = 16). Phenylglyoxal (10 mM), an arginine modifying reagent, reduced the maximum amount of charge movement to 14% of control. The inhibitory effect of phenylglyoxal was time dependent and the decline time course of maximum amount of charge movement could be fitted by a single exponential curve with a time constant of 5.79 min. The dihydropyridine (DHP) receptor antagonist, nifedipine, immobilized 54% of the charge movement. These results suggest that a part of the charge movement reflects the conformational change of the DHP receptors upon membrane depolarization.