J. L. Marin

Folding–unfolding of FN-III domains in tenascin: an elastically coupled two-state system

In a single-molecule atomic force microscopy (AFM) experiment, the tenascin molecule is stretched by an external force causing an elongation which is due to the unfolding of the FN-III modules. The features of the force-extension curves depend on the pulling speed and show a saw-tooth pattern (lower speeds) or a smooth pattern (higher speeds). In any case, the unfolded domains are elastically coupled to the unfolded modules, acting as transmitters of the external force. In this communication, the folding-unfolding process of the FN-III domains in tenascin is studied using reaction rate theory and a simple two-state model. The main hypothesis of the study is that, at microscopic level, the force needed to unfold a domain and the unfolding rate (unfolding velocity) can mimic the macroscopic process of measurement by AFM. As the external force is applied, the probability of unfolding increases as dictated by the reaction rate theory. Within this context, a relationship between the unfolding force and the unfolding velocity is obtained. The latter relation will describe microscopically the process in a phenomenological fashion. Moreover, while relating the results of this study with other experimental (AFM measurements) and theoretical (Monte Carlo simulations) data, we found that the graph of unfolding force-unfolding velocity is similar to that of external force-pulling velocity. The refolding process can also be studied within this model and the results show similar trends. The latter suggests a generic and universal behavior of such kind of molecular domains at least in the light of the proposed model.

Measurement of Ca2+ currents in intact slow skeletal muscle fibers of the frog by the three-microelectrode technique

The recording of currents passing through calcium channels in intact skeletal muscle fibers presents several difficulties. However, use of the three-microelectrode voltage-clamp technique at the end of the fiber provided us with a good approximation of current values in such fibers. Using this technique, we were able to measure the calcium-channel current in slow skeletal muscle fibers of the frog (Rana pipiens) and to quantify the effects of denervation on this current.

Sodium Withdrawal Contractures in Tonic Skeletal Muscle Fibers of the Froga

Skeletal muscle possesses two different fibers: twitch and tonic or slow. Tonic fibers, in contrast to twitch fibers, can maintain tension during prolonged depolarization.1.2 The sodium-calcium exchange system was investigated by contractile response to alteration of the extracellular sodium concentration in the tonic muscle fibers of the frog. Isometric tension was recorded from tonic bundles of cruralis muscle of Rana pipiens. Normal solution was (mM): NaCl 117.5, KC12.5, CaCl, 1.8; pH was adjusted to 7.4 with imidazole-chloride. Na+-free solutions were prepared by replacing NaCl with an osmotically equivalent amount of Tris-C1, TMA-Cl, TEA-Cl or N-methylglucamine-C1. In some experiments 15 pM of veratridine or 1 pM of strophantidin was added to normal solution. When Ca2+ was omitted from the solutions, it was replaced by 0.5 mM NiClP3 All experimental solutions contained d-tubocurarine (50 pM) to prevent a possible acetylcholine-like effect.* Experiments were done at room temperature (20-22 C). Contractures were evoked in solutions where the extracellular sodium was withdrawn. FIGURE 1 is a representative result of the effect of sodiumwithdrawal solution containing d-tubocurarine. The initial tension developed slowly, reaching a maximum to establish a plateau. On return to normal solution, there was a second rise in tension that relaxed spontaneously. This second response was somewhat variable and depends upon the compounds that substitute NaC1. Similar results were obtained when sodium was substituted with Tris, TMA, or glucamine. The replacement of NaC by TEA+ did not affect the resting potential. It was -77 2 8 mV (n = 10) in normal solution and -72 f 6 mV (n = 9) in TEA-CI solution. In the presence of d-tubocurarine, the amplitude of sodium-withdrawal contracture was diminished, and its time course was modified. However, when Na+ was replaced by TEA+ the sodiumwithdrawal contractures were not modified by the d-tubocurarine. Contracture induced in Na+ withdrawal are highly dependent on external Ca2+ concentration. If external calcium was omitted from the solution, the tension of sodium-withdrawal contracture was greatly reduced (FIG. 2, striped bar) as compared to control (FIG. 2, left empty bar), and it was re-established once external calcium was restored (FIG. 2, right empty bar). On the other hand, veratridine and strophantidin tend to increase the intracellular sodium concentration. If the bundles were soaked in normal solution with these drugs, added 20 min before the~ontracture, the tension was increased. The present results

Further analysis of the molecular jet hypothesis during muscle contraction

We analyse the Molecular Jet-hypothesis proposed by Morel & Bachouchi (1988, J. theor. Biol. 132, 83.). This hypothesis attempts to explain the movement of covaspheres that contain myosin heads attached to actin filaments. This movement occurs during muscle contraction. However, the hypothesis does not predict the velocity of covaspheres correctly. Therefore, we are studying additional aspects of the hypothesis.

Effects of denervation on Ca2+ channels in slow skeletal muscle fibers of the frog.

Effects of denervation on calcium channels in slow skeletal muscle fibers in the frog (Rana pipiens) were studied using the three-microelectrode voltage-clamp technique in intact fibers. Ca2+, Ba2+, and Sr2+ currents were all significantly reduced in amplitude during the first 2 weeks after denervation. After nerve section the selectivity sequence Ba congruent with Ca > Sr was changed to Ba > Sr > Ca and the values for relative ratio increased from 1.04 to 2.65 for Ba2+ and from 0.58 to 1.20 for Sr2+ (with respect to Ca2+). Barium current saturation was more obvious in denervated fibers than in non-denervated fibers. The values obtained with the Michaelis-Menten type expression, I = Imax/(1+Kd/[Ba]e) were Kd = 2.7 mM and Imax = 20 microA/cm2 in fibers 2 weeks after nerve section compared with the values Kd = 4.4 mM and Imax = 60 microA/cm2 obtained in non-denervated fibers. Additionally, the effects of two calcium channel blockers (cobalt and nifedipine) were greater by a factor of two in denervated fibers than in non-denervated fibers. Three weeks or so after nerve section, all the biophysical properties studied began to show a tendency to recover toward the values obtained in non-denervated muscles (controls). These results suggest that calcium channels are modified or that there is a change in the types of calcium channels present in frog slow skeletal muscle fibers after denervation.