Calcium and strontium concentration changes within skinned muscle preparations following a change in the external bathing solution.

230

152

SUMMARY1. Steady-state force-pCa relations were determined for mechanically skinned fibres of fast-and slow-twitch rat skeletal muscle, extensor digitorum longus (e.d.l.) and soleus respectively, at varied sarcomere lengths and temperatures (3-35 'C).2. This relation was different for the two fibre types at all sarcomere lengths, with soleus fibres having lower Ca2+ concentration threshold for activation and requiring a wider Ca2+ concentration range to reach force saturation.3. An increase in sarcomere length within the range 2-2-3-6 ,um reversibly shifted the steady-state force-pCa curves for both fast-and slow-twitch fibres toward lower Ca2+ without affecting the steepness of the curves.4. The results are consistent with the idea that the sensitivity of the contractile apparatus to Ca2+ is increased with increasing sarcomere length.5. The change in apparent Ca2+ sensitivity (measured as the change in pCa corresponding to 50 % Ca-activated force (ApCa50)) was approximately proportional to the change in sarcomere length (ASL) at all temperatures for both types of muscle fibre.6. The proportionality coefficient (ApCa50/ASL) was higher for slow-twitch fibres than for fast-twitch fibres by a factor of 1P5-2-0 at all temperatures investigated.7. The force-pCa relation was less affected by changes in sarcomere length at lower (3-5 'C) than at higher temperatures (22-35 'C).8. The sarcomere length-maximum force relation obtained with skinned fibres for sarcomere lengths between 2 10 and 4 05 jsm was similar for both fibre types at all temperatures despite large temperature dependent variations in maximum Caactivated force response. Maximum force decreased linearly to zero between ca. 2-8 and 3.95 /sm; remained high and essentially constant between ca. 2-40 and 2-80 him, and decreased significantly below 2-40 ,um.9. Myofilament-generated tension oscillations produced by partially activated soleus skinned fibres are highly length dependent, disappearing completely when sarcomere length is increased above 3-15 /sm.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Effects of sarcomere length on the force—pCa relation in fast‐ and slow‐twitch skinned muscle fibres from the rat

Stephenson¹,

Williams²

1982

230

152

“…Note that the ability of the contractile apparatus to develop maximum Ca¥-activated force decreases only marginally for the duration of an experiment. Control experiments with three freshly dissected mechanically skinned muscle fibres activated successively by rapidly raising [Ca¥] to 4 ìÒ and then decreasing it to below 0·1 ìÒ (Moisescu, 1976;Moisescu & Thieleczek, 1978) have shown that the maximal Ca¥-activated force obtained at 30 ìÒ [Ca¥] decreased by only 6·6 ± 0·2 % after 35 activation-relaxation cycles lasting 4-6 s where the average force response was 85·5 ± 3·0 % of the maximum Ca¥-activated force. Both the duration of the activation and the force level attained during activation were considerably higher than the corresponding averages for the depolarization-induced force responses to 50% run-down.…”

Section: T_system Depolarization Experimentsmentioning

confidence: 99%

Glycogen content and excitation‐contraction coupling in mechanically skinned muscle fibres of the cane toad

Stephenson

Nguyễn

Stephenson

1999

There are consistent observations that depletion of intracellular glycogen stores in muscle is associated with reduced muscle performance (Bergstr om et al. 1967;Galbo et al. 1979;Voellestad et al. 1988;Fitts, 1994;Chin & Allen, 1997), but the cellular mechanisms responsible for the glycogen depletion-related impairment of muscle function are not understood (Fitts 1994;Chin & Allen, 1997). So far, all studies on the role of glycogen in muscle have involved the depletion of the muscle glycogen pool by inducing a state of muscle fatigue. Therefore, from these studies it is not possible to draw unequivocal conclusions concerning the nature of the relationship between glycogen content and muscle contractility because in addition to glycogen depletion, many other factors, known to affect excitation-contraction (E-C) coupling (Stephenson et al. 1998), would have been altered in the myoplasm of the fatigued muscle fibres. Further advances can be made if the relationship between glycogen and muscle contractility is investigated in single fibres containing different concentrations of glycogen under conditions where the ionic composition of the myoplasmic environment is kept constant and similar to that in a rested muscle. The mechanically skinned single muscle fibre preparation is particularly well suited for such a task

“…For the extracellular solutions, the 10 mÒ BAPTA was replaced by 8 mÒ EGTA and 2 mÒ EDTA (0 Mg¥ control solution) or 10 mÒ EGTA to which MgClµ was added to yield the free Mg¥ concentration indicated in the text. Free Mg¥ concentrations were determined from apparent affinity constants for EGTA, measured with the potentiometric method, with software kindly provided by R. Thieleczek (Moisescu & Thieleczek, 1978;Stephenson & Thieleczek, 1986). In all instances block was measured using whole-cell recordings of HEK 293 cells.…”

Section: Solutionsmentioning

confidence: 99%

Adjacent asparagines in the NR2‐subunit of the NMDA receptor channel control the voltage‐dependent block by extracellular Mg²⁺

Wollmuth

Kuner

Sakmann

1998

138

159

1. The voltage-dependent block of N-methyl-ª_aspartate (NMDA) receptor channels by extracellular Mg¥ is a critical determinant of its contribution to CNS synaptic physiology. The function of the narrow constriction of the channel in determining the block was investigated by analysing the effects of a set of different amino acid substitutions at exposed residues positioned at or near this region. NMDA receptor channels, composed of wild-type and mutant NR1-and NR2A-subunits, were expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. 2. In wild-type channels, the voltage dependence (ä) of the block by Mg¥ was concentration dependent with values of ä of •0·58 in 0·01 mÒ and •0·82 in 0·07 mÒ and higher concentrations. Under biionic conditions with high extracellular Mg¥ and K¤ as the reference ion, Mg¥ weakly permeated the channel. Over intermediate potentials (•−60 to −10 mV), this weak permeability had no apparent effect on the block but at potentials negative to •−60 mV, it attenuated the extent and voltage dependence of the block. 3. Substitutions of glycine, serine, glutamine or aspartate for the N-site asparagine in the NR1-subunit enhanced the extent of block over intermediate potentials but left the voltage dependence of the block unchanged indicating that structural determinants of the block remained. These same substitutions either attenuated or left unchanged the apparent Mg¥ permeability. 4. In channels containing substitutions of glycine, serine or glutamine for the N-site asparagine in the NR2A-subunit, the block by Mg¥ was reduced at negative potentials. Over intermediate potentials, the block was not strongly attenuated except for the glutamine substitution which reduced the voltage dependence of the block to •0·57 in 0·7 mÒ Mg¥. 5. Equivalent substitutions for the N + 1 site asparagine in the NR2A-subunit strongly attenuated the block over the entire voltage range. In 0·7 mÒ Mg¥, the voltage dependence of the block was reduced to 0·50 (glycine), 0·53 (serine) and 0·46 (glutamine). 6. Channels containing substitutions of the N-site or N + 1 site asparagines in the NR2A-subunit showed an increased Mg¥ permeability suggesting that these adjacent asparagines form a barrier for inward Mg¥ flux. Changes in this barrier contribute, at least in part, to the mechanism underlying disruption of the block following substitution of these residues. 7. The adjacent NR2A-subunit asparagines are positioned at or near the narrow constriction of the channel. Pore size, however, did not determine how effectively Mg¥ blocks mutant channels. 8. It is concluded that, at the narrow constriction in the NMDA receptor channel, the adjacent NR2A-subunit asparagines, the N-site and N + 1 site, but not the N-site asparagine of the NR1-subunit, form a critical blocking site for extracellular Mg¥. The contribution to the blocking site, in contrast to the prevailing view, is stronger for the N + 1 site than for the N_site asparagine. The block may involve binding of Mg¥ to these residues.