Juan José Marengo

Activation of calcium channels in sarcoplasmic reticulum from frog muscle by nanomolar concentrations of ryanodine.

Sarcoplasmic reticulum vesicles isolated from fast-twitch frog skeletal muscle presented two classes of binding sites for ryanodine, one of high affinity (Kd1 = 1.7 nM, Bmax1 = 3.3 pmol per mg) and a second class with lower affinity (Kd2 = 90 nM, Bmax2 = 7.0 pmol per milligram). The calcium channels present in the sarcoplasmic reticulum membranes were studied in vesicles fused into lipid bilayers. Low concentrations of ryanodine (5 to 10 nM) activated a large conductance calcium channel after a short delay (5 to 10 min). The activation, which could be elicited from conditions of high or low fractional open time, was characterized by an increase in channel fractional open time without a change in conductance. The open and closed dwell time distributions were fitted with the sum of two exponentials in the range of 4 to 800 ms. The activating effect of ryanodine was due to an increase of both open time constants and a concomitant decrease in the closed time constants. Under conditions of low fractional open time (less than 0.1), the time spent in long closed periods (greater than 800 ms) between bursts was not affected by ryanodine. Higher concentrations of ryanodine (250 nM) locked the channel in a lower conductance level (approximately 40%) with a fractional open time near unity. These results suggest that the activating effects of nanomolar concentrations of ryanodine may arise from drug binding to high affinity sites. The expression of the lower conductance state obtained with higher concentrations of ryanodine may be associated with the low affinity binding sites observed in frog sarcoplasmic reticulum.

Sarcoplasmic reticulum release channels from frog skeletal muscle display two types of calcium dependence

Calcium channels derived from sarcoplasmic reticulum of frog skeletal muscle were fused with planar lipid bilayers. Fractional open times displayed two types of calcium dependence: (i) blockable channels showed a bell‐shaped calcium dependence with an activation constant of 4.5 μM, a Hill coefficient for activation of 1.46 and a blocking constant of 226 μM, and (ii) non‐blockable channels displayed a sigmoidal calcium dependence with an activation constant of 1.1 μM and a Hill coefficient of 1.42; no blocking effect was seen with calcium up to 0.5 mM. These two types of calcium dependence may underlie the coexistence of two different pathways for calcium release in frog skeletal muscle.

Fetal brain hypometabolism during prolonged hypoxaemia in the llama

In this study we looked for additional evidence to support the hypothesis that fetal llama reacts to hypoxaemia with adaptive brain hypometabolism. We determined fetal llama brain temperature, Na+ and K+ channel density and Na+–K+‐ATPase activity. Additionally, we looked to see whether there were signs of cell death in the brain cortex of llama fetuses submitted to prolonged hypoxaemia. Ten fetal llamas were instrumented under general anaesthesia to measure pH, arterial blood gases, mean arterial pressure, heart rate, and brain and core temperatures. Measurements were made 1 h before and every hour during 24 h of hypoxaemia (n= 5), which was imposed by reducing maternal inspired oxygen fraction to reach a fetal arterial partial pressure of oxygen (P  a,O 2 ) of about 12 mmHg. A normoxaemic group was the control (n= 5). After 24 h of hypoxaemia, we determined brain cortex Na+–K+‐ATPase activity, ouabain binding, and the expression of NaV1.1, NaV1.2, NaV1.3, NaV1.6, TREK1, TRAAK and KATP channels. The lack of brain cortex damage was assessed as poly ADP‐ribose polymerase (PARP) proteolysis. We found a mean decrease of 0.56°C in brain cortex temperature during prolonged hypoxaemia, which was accompanied by a 51% decrease in brain cortex Na+–K+‐ATPase activity, and by a 44% decrease in protein content of NaV1.1, a voltage‐gated Na+ channel. These changes occurred in absence of changes in PARP protein degradation, suggesting that the cell death of the brain was not enhanced in the fetal llama during hypoxaemia. Taken together, these results provide further evidence to support the hypothesis that the fetal llama responds to prolonged hypoxaemia with adaptive brain hypometabolism, partly mediated by decreases in Na+–K+‐ATPase activity and expression of NaV channels.

Calcium dependence of ryanodine-sensitive calcium channels from brain cortex endoplasmic reticulum.

Endoplasmic reticulum vesicles isolated from rat brain cortex and fused with lipid bilayers displayed ryanodine‐sensitive calcium channels, with three cytoplasmic calcium dependences. A: Channels (n = 5) stimulated by Ca2+ (K 0.5 = 1.2 μM and n Hill = 1.9) and not inhibited up to 0.5 mM Ca2+. B: Channels (n = 14) cooperatively activated (K 0.5 = 6.9 μM and n Hill = 1.8), and inhibited by Ca2+ (K 0.5 = 152 μM and n Hill = 1.8). C: Low P o (< 0.1) channels (n = 22), non‐cooperatively activated and inhibited with the same K 0.5 = 26.3 μM Ca2+. These three types of responses to cytoplasmic [Ca2+] may underlie separate calcium release pathways in neurons of rat brain cortex.

Activation of inositol trisphosphate‐sensitive Ca2+ channels of sarcoplasmic reticulum from frog skeletal muscle.

1. The modulation by Ca2+ of the activation by inositol 1,4,5‐trisphosphate (IP3) of Ca2+ channels present in native sarcoplasmic reticulum membranes from frog skeletal muscle was studied after channel incorporation into planar phospholipid bilayers in the presence of Ca2+ or Ba2+ as current carrier species. 2. Channel activity expressed as fractional open time (Po) was low (less than or equal to 0.15) in the presence of varying free Ca2+ concentrations bathing the myoplasmic face of the channel (cis side), and did not increase significantly between 0.01 and 30 microM‐Ca2+. 3. Channel activation mediated by IP3 could be elicited from free Ca2+ levels similar to those of resting skeletal muscle (about 0.1 microM) and was found to be strongly regulated by the free Ca2+ concentration present at the myoplasmic moiety of the channel. 4. Channel activation by 10 microM‐IP3 depended on the Ca2+ concentration on the cis side. Po reached a maximum between pCa 7.0 and 6.0, but decreased at higher concentrations of free Ca2+. Thus, Ca2+ exerted a modulatory influence on IP3‐mediated activation in a concentration range where the channel was insensitive to Ca2+. 5. The results indicate that Ca2+ ions act as modulators of IP3 efficacy to open the channel. This could arise from an interaction of Ca2+ with the channel gating mechanism or with the agonist binding site.

Antagonistic effects of TrkB and p75NTR on NMDA receptor currents in post‐synaptic densities transplanted into Xenopus oocytes

Brain‐derived neurotrophic factor (BDNF) and its receptor TrkB are essential regulators of synaptic function in the adult CNS. A TrkB‐mediated effect at excitatory synapses is enhancement of NMDA receptor (NMDA‐R)‐mediated currents. Recently, opposing effects of TrkB and the pan‐neurotrophin receptor p75NTR on long‐term synaptic depression and long‐term potentiation have been reported in the hippocampus. To further study the regulation of NMDA‐Rs by neurotrophin receptors in their native protein environment, we micro‐transplanted rat forebrain post‐synaptic densities (PSDs) into Xenopus oocytes. One‐minute incubations of oocytes with BDNF led to dual effects on NMDA‐R currents: either TrkB‐dependent potentiation or TrkB‐independent inhibition were observed. Pro‐nerve growth factor, a ligand for p75NTR but not for TrkB, produced a reversible, dose‐dependent, TrkB‐independent and p75NTR‐dependent inhibition of NMDA‐Rs. Fractionation experiments showed that p75NTR is highly enriched in the PSD protein fraction. Immunoprecipitation and pull‐down experiments further revealed that p75NTR is a core component of the PSD, where it interacts with the PDZ3 domain of the scaffolding protein SAP90/PSD‐95. Our data provide striking evidence for a rapid inhibitory effect of p75NTR on NMDA‐R currents that antagonizes TrkB‐mediated NMDA‐R potentiation. These opposing mechanisms might be present in a large proportion of forebrain synapses and may contribute importantly to synaptic plasticity.

Calcium Channels in Sarcoplasmic Reticulum Membranes Isolated from Skeletal Muscle

Calcium channels have been detected in sarcoplasmic reticulum (SR) membranes isolated from rabbit(1,2) and frog(3–5) skeletal muscle. Several lines of evidence indicate that these conductances participate in calcium release during excitation-contraction coupling(1–6) High-conductance channels present in SR isolated from rabbit(1) and frog SR muscle(3–5) are activated by ATP and by calcium applied at the myoplasmic side, and are blocked by magnesium and ruthenium red(1,6) The channel present in SR membranes from frog is activated by micromolar concentrations of 1,4,5-inositol tri-sphosphate(4,5) a postulated internal agonist of excitation-contraction coupling. (7–10) This drug increases fractional open time (P 0) in a concentration-dependent manner without an effect on single-channel conductance. Gating and conductance of the same channel are modified by nanomolar concentrations of ryanodine(3,11) a plant alkaloid that elicits irreversible muscle contractures.