François Tiaho

Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy.

We performed Second Harmonic Generation (SHG) imaging microscopy of endogeneous myosin-rich and collagen-rich tissues in amphibian and mammals. We determined the relative components of the macroscopic susceptibility tensor chi((2)) from polarization dependence of SHG intensity. The effective orientation angle theta(e) of the harmonophores has been determined for each protein. For myosin we found theta(e) approximately 62 degrees and this value was unchanged during myofibrillogenesis. It was also independent of the animal species (xenopus, dog and human). For collagen we found theta(e) approximately 49 degrees for both type I- and type III- rich tissues. From these results we localized the source of SHG along the single helix of both myosin and collagen.

Regulation of the frequency‐dependent facilitation of L‐type Ca2+ currents in rat ventricular myocytes.

1. An increase in the rate of stimulation induces an augmentation of L‐type Ca2+ currents (ICa) and concomitant slowing of current decay in rat ventricular cells. This facilitation is quasi immediate (1‐3 s), graded with the rate of stimulation, and occurs only from negative holding potentials. We investigated this effect using trains of stimulation at 1 Hz and the whole‐cell patch‐clamp technique (18‐22 degrees C). 2. The decay of ICa is normally bi‐exponential and comprises fast and slow current components (ICa,fc and ICa,sc, respectively). Facilitation of ICa was observed only when ICa,fc was predominant. 3. Facilitation developed during the run‐up of ICa with the interconversion of ICa,sc into ICa,fc, and vanished during the run‐down of ICa with the loss of ICa,fc.Ni2+ (300 microM) and nifedipine (1 microM) suppressed facilitation owing to the preferential inhibition of ICa,fc. 4. Facilitation of ICa was not altered (when present) or favoured (when absent) by the cAMP‐dependent phosphorylation of Ca2+ channels promoted by isoprenaline or by intracellular application of cAMP or of the catalytic subunit of protein kinase A (C‐sub). A similar effect was observed when the dihydropyridine agonist Bay K 8644 was applied. In both cases, facilitation was linked to a preferential increase of ICa,fc. 5. Following intracellular application of inhibitors of protein kinase A in combination with a non‐hydrolysable ATP analogue, ICa consisted predominantly of ICa,sc and no facilitation was observed. The calmodulin antagonist naphthalenesulphonamide had no effect on facilitation. 6. When Bay K 8644 was applied in combination with isoprenaline, cAMP or C‐sub, the decay of ICa was slowed with the predominant development of ICa,sc, and facilitation of ICa was nearly abolished. Facilitation also depended on extracellular Ca2+, and was suppressed when Ba2+ replaced Ca2+ as the permeating ion. 7. When no EGTA was included in the patch pipette, facilitation was not further enhanced but a use‐dependent decrease of ICa frequently occurred. When BAPTA was used in place of EGTA, the rate of inactivation of ICa was reduced and facilitation was abolished. 8. In conclusion, the facilitation of ICa that reflects a voltage‐driven interconversion of ICa,fc into ICa,sc is also regulated by Ca2+ and by cAMP‐dependent phosphorylation. The presence of the gating pattern typified by ICa,fc is required. Ca2+ may exert its effect near the inner pore of the Ca2+ channel protein and control the distribution between the closed states of the two gating pathways.

Cyclic-AMP-dependent phosphorylation modulates the stereospecific activation of cardiac Ca channels by Bay K 8644

Voltage-gated Ca channels have been reported to be regulated by membrane potential, phosphorylation and binding of specific agonists or antagonists such as dihydropyridines. We report here evidence that cyclic AMP (cAMP) modulates the activation of Ca-channel current by the dihydropyridine agonist Bay K 8644. Bay K 8644 (racemate) alone induces a primary voltage-dependent, potentiating effect on peak current amplitude and accelerates the current decay. In contrast, in the presence of cAMP activators, we observed a striking slowing of the decay in addition to the increase in peak current. The agonist (−)-Bay K 8644, but not the antagonist (+)-Bay K 8644, when applied in combination with cAMP, forskolin or isoproterenol, mimics the effect of the racemate. We have interpreted the results presented here in respect of a cAMP-dependent modulation of Bay K 8644 effects on cardiac Ca-channel currents. It may open the new perspective that dephosphorylated and phosphorylated Ca channels have distinct pharmacology.

Voltage-dependent regulation of L-type cardiac Ca channels by isoproterenol

The β-adrenergic cascade is important for the regulation of voltage-dependent Ca channels by phosphorylation. Here we report that isoproterenol (ISO) profoundly alters the voltage-dependent properties of L-type Ca channels studied in rat ventricular cells. ISO (1 μM) shifted both threshold and maximal activation of Ba current (IBa) towards more negative potentials (approx. 10 mV). An equivalent shift was observed in the steady-state voltage-dependent inactivation curve. As a consequence, the potentiation induced by ISO on IBa was greater for weak depolarizations and from negative holding potentials (Vh). We have excluded that the contribution of minor uncompensated series resistances, the activation of Cl currents or changes in junction potential during the experiments account for these effects. In addition, ISO had a dual effect on IBa decay depending on the voltage step (acceleration below, slowing above −10 mV). In conclusion, it is postulated that the voltage dependence of the potentiating effects of ISO on Ca channels activity may ensure a selective regulation among heart tissues with different membrane resting potentials.

Characterization of voltage‐dependent calcium channels expressed in Xenopus oocytes injected with mRNA from rat heart.

1. The properties of voltage dependent cardiac Ca channels expressed in Xenopus laevis oocytes after injection of mRNA from rat heart were investigated using the double‐microelectrode voltage‐clamp technique. 2. Endogenous Ba current (IBa,E) and expressed cardiac Ba current (IBa,C) were studied at various external concentrations of barium (Ba2+). These two entities could be distinguished by their amplitude and their pharmacology. IBa,C was more sensitive to the inorganic Ca channel blocker manganese (Mn2+). The contaminant IBa,E presented properties of voltage dependence identical to IBa,C, but was negligible in the presence of a low external Ba2+ concentration (2 mM). 3. In 2 mM‐Ba2+, IBa,C activated at ‐35 mV, peaked at ‐14 mV, and reversed at +26 mV. Steady‐state inactivation properties, in consideration of the half‐inactivation potential of ‐35 mV, were also typical of L‐type Ba currents. However, the decay of IBa,C was very slow (time constant of inactivation near 600 ms). No evidence for the expression of cardiac transient Ca channels (T‐type) was found. 4. IBa,C was enhanced after exposure to the 1,4‐dihydropyridine (DHP) agonist Bay K 8644. The enhancement of IBa,C was voltage dependent (maximum at ‐30 +/‐ 5 mV) and associated with a slowing in current decay. Current‐voltage and concentration‐response curves obtained for various Ba2+ concentrations revealed an antagonism between external Ba2+ and the 1,4‐DHP agonist Bay K 8644. Similar results were found using the (‐)Bay K 8644 pure agonist isomer. 5. We conclude that oocytes injected with mRNA from rat heart expressed only the high threshold, long‐lasting or L‐type Ca channels. The availability of expressed L‐type Ca channels for quantitative pharmacological studies using low Ba2+ concentration has been demonstrated.

Three distinct sarcomeric patterns of skeletal muscle revealed by SHG and TPEF Microscopy

We have extensively characterized the sarcomeric SHG signal as a function of animal species (rat versus xenopus), age (adult versus larval) and tissue preparation (fixed or fresh) and we found that the main feature of this signal is a single peak per mature sarcomere (about 85% of all sarcomeres). The remaining (15%) was found to be either double peak per mature sarcomere or mini sarcomeres (half of a sarcomere) using alpha-actinin immuno detection of the Z-band. The mini sarcomeres are often found in region of pitchfork-like SHG pattern. We suggest that double peak SHG pattern could indicate regions of sarcomeric proteolysis whereas pitchfork-like SHG pattern could reveal sarcomeric assembly.

Repriming of L‐type calcium currents revealed during early whole‐cell patch‐clamp recordings in rat ventricular cells.

1. The establishment of the whole‐cell patch‐clamp recording configuration (WCR) revealed a type of inhibition to which L‐type Ca2+ channels were subject in static rat ventricular myocytes before obtaining the WCR. 2. Immediately after membrane disruption (< 10 s), the Ca2+ current (ICa) was absent but gradually increased in amplitude to reach its final waveform (amplitude and kinetics) 2‐3 min after the WCR was reached. 3. Three distinct phases (P) were identified. First, no inward but an outward current, blocked (1‐2 min) by Cs+ dialysing from the patch pipette (P1), was recorded. Second, overlapping with (P1), ICa increased dramatically to reach a maximum peak amplitude within 2‐3 min (P2). Concomitantly, its rate of decay, initially monoexponential and slow, became biexponential owing to the appearance of a fast component of inactivation (P3). Complete interconversion between slow and fast components sometimes occurred. 4. Changes in current waveform were not related to voltage loss or series resistance variation, and suppression of an outward current (P1) was unlikely to account for P2 and P3. 5. The run‐up of ICa was independent of the nature of the permeating ions, the membrane holding potential, depolarization, rate of stimulation, the intracellular Ca2+, ATP, Mg2+, Cs+ and the pH of the pipette solution. Since large Ca2+ currents were recorded using the perforated patch technique, the run‐up of ICa is not explained by the wash‐out of an inhibitory endogenous macromolecule during cell‐pipette exchanges. 6. Pharmacological manipulations, including the use of Ca(2+)‐Ba(2+)‐EGTA and exposure of the cells to isoprenaline and/or Bay K 8644 prior to recording, did not alter the mechanism primarily responsible for build‐up. Unrepriming of channel activity was required before these modulations could be effective. 7. Currents could however be instantly augmented when cells were extracellularly superfused during the run‐up step. The wash‐out of an inhibitory agent originating in the cell itself (such as H+, NH4+ and lactate) and accumulating in the extracellular microenvironment of the cells seems unlikely. Rather, we suggest that pressure‐induced mechanostimulation may be involved in the restoration of Ca2+ channel activity.

Some aspects of the physiological role of ion channels in the nervous system

Recent analyses of the genomes of several animal species, including man, have revealed that a large number of ion channels are present in the nervous system. Our understanding of the physiological role of these channels in the nervous system has followed the evolution of biophysical techniques during the last century. The observation and the quantification of the electrical events associated with the operation of the ionic channels has been, and still is, one of the best tools to analyse the various aspects of their contribution to nerve function. For this reason, we have chosen to use electrophysiological recordings to illustrate some of the main functions of these channels. The properties and the roles of Na+ and K+ channels in neuronal resting and action potentials are illustrated in the case of the giant axons of the squid and the cockroach. The nature and role of the calcium currents in the bursting behaviour of the neurons are illustrated for Aplysia giant neurons. The relationship between presynaptic calcium currents and synaptic transmission is shown for the squid giant synapse. The involvement of calcium channels in survival and neurite outgrowth of cultured neurons is exemplified using embryonic cockroach brain neurons. This same neuronal preparation is used to illustrate ion channel noise and single-channel events associated with the binding of agonists to nicotinic receptors. Some features of the synaptic activity in the central nervous system are shown, with examples from the cercal nerve giant-axon preparation of the cockroach. The interplay of different ion conductances involved in the oscillatory behaviour of the Xenopus spinal motoneurons is illustrated and discussed. The last part of this review deals with ionic homeostasis in the brain and the function of glial cells, with examples from Necturus and squids.

Effects of nicotinic and muscarinic ligands on embryonic neurones of Periplaneta americana in primary culture: a whole cell clamp study

The pharmacological properties of acetylcholine (ACh) receptors of cultured neurones from embryonic cockroach brains were studied using the whole-cell configuration of the patch-clamp technique. More than 90% of the studied neurones responded to ACh by a monophasic inward current, the intensity of which varied from cell to cell. The sequence of potency of the five tested agonists was ACh > nicotine=carbamylcholine > suberyldicholine=oxotremorine. The dose-response relationship was complex, suggesting the existence of two populations of receptors: high-affinity receptors (extrapolated K(d) around 10(-7) M) and low-affinity receptors (extrapolated K(d) around 5x10(-5) M). The current-voltage relationship of the induced current was linear between -80 and -40 mV and the extrapolated reversal potential was not significantly different from 0 mV. The sequence of decreasing potency of the antagonists of the ACh response was: methyllycaconitine > alpha-bungarotoxin > mecamylamine > curare > strychnine > bicuculline > atropine > picrotoxin. These results show: (1) that, in embryonic brain neurones, the response to ACh corresponds to the opening of non-selective cationic channels; and (2) that the pharmacology of the ACh receptors is mainly but not solely nicotinic. The nature of the single events which underlie this response, as well as the structure of the channels (homo or hetero-oligomeric) remain to be investigated.

Double-band sarcomeric SHG pattern induced by adult skeletal muscles alteration during myofibrils preparation.

To understand the reported difference between double band, sarcomeric second harmonic generation pattern of isolated myofibril and predominant single band pattern found in thick muscle tissues, we studied the effect of myofibril preparation on the second harmonic generation pattern. We found that double band sarcomeric second harmonic generation pattern usually observed in myofibrils (prepared from fresh tissue) is due to muscle alteration during the mixing and triton treatment processes. Single band sarcomeric second harmonic generation pattern could be observed in isolated myofibrils when this alteration is previously prevented using paraformaldehyd fixed tissue. We conclude that single band sarcomeric second harmonic generation pattern is a signature of adult muscle myofibrils in normal physiological condition, suggesting that sarcomeric second harmonic generation patterns could be used as a valuable diagnosis tool of muscle health.