The relationship between tension and Ca2+ concentration in single skinned muscle fibers has been determined with a high density of experimental points and the data have been fitted by a least squares method to the Hill equation. We find that the mean Hill coefficient for the slope of the tension/Ca2+ relationship is between 5 and 6, and the pKld is about 5.9. Because there are four Ca2+ binding sites on troponin C, and only two of these regulate hydrolysis of MgATP, we conclude that the regulation of tension by Ca2+ binding is greatly modified by other factors. One important factor is the time required for a cross-bridge to complete a cycle once initiated, relative to the time Ca2+ remains bound to troponin C. The pCa/tension relationship will shift to higher pCa values as the ratio of cross-bridge cycle time to the Ca2+ bound time increases. For example, the pCa/tension curve may progressively shift to the left with increase in tension because strain in the myofilament lattice progressively increases the cycle time. This left shift will produce a ta/tension relationship that is steeper than the actual Ca2 binding curve. The anticipated shift of the pCa/ tension curve with cycle time also bears on interpretations of earlier experiments on the "active state" and on the effects of Ca2+ on the maximal velocity of shortening.

Three different ways of shifting the pCa/tension curve on the pCa axis have been studied and related to changes in the rate constants of the crossbridge cycle. The curve midpoint shifts to higher pCa's wheel the substrate (Mg-ATP) is reduced from 5 to 0.25 mM, when the phosphate concentration is reduced from 7.5 mM to 0, and when the ionic strength is reduced from 0.200 to 0.120 . The Hill coefficients of the pCa/tension curve in our standard saline (5 mM substrate, 5 mM free ATP, 7.5 mM phosphate, ionic strength 0.200, 15°C) are between 5.1 and 5.6 and fall to 3.0 with the left shift of the curve brought about by reducing both substrate and phosphate. Left shifts of the curve produced by reduction in the ionic strength do not result in a lower Hill coefficient . Reducing either substrate or phosphate is associated with a reduction in the optimal frequency for oscillatory work, but reduction in ionic strength is not so associated . Maximum tension increases with the left shift of the curve brought about by reducing phosphate concentration or ionic strength, but tension decreases with the left shift of the curve accompanying substrate concentration reduction in a phosphate-free saline . We argue that one mechanism for the observed shift of the curve along the pCa axis is the relationship between the time a cross-bridge takes to complete a cycle and the time Ca 2+ stays bound to troponin C (TnC) . If the cycle rate is decreased, a smaller fraction of TnC sites must be occupied to keep a given fraction of cross-bridges active . To illustrate this concept, we present a simplified model of the cross-bridge cycle incorporating the kinetics of Ca binding to TnC.

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The nicotinic acetylcholine receptor is a multisubunit, membrane-spanning protein that contains a gated, cation-conducting channel. Our approach to the understanding of the function of this receptor in molecular terms has been to locate its functionally significant sites in the sequences of its subunits and in its three-dimensional structure. In addition, we have tried to correlate transitions in the properties of these sites with functional transitions of the receptor. On binding acetylcholine, the nicotinic acetylcholine receptor enters at least two transient states, the open state and the rapid-onset desensitized state, and, in the continued presence of agonist, finally subsides into the slow-onset desensitized state. The transitions of the receptor between these various states are susceptible to regulation by acetylcholine and its congeners acting at one type of site and by a broad class of noncompetitive inhibitors (NCIs), including local anesthetics, acting at other sites. The chain composition of the receptor is alpha 2 beta gamma delta. The two acetylcholine binding sites are on the alpha chains, and two residues contributing to these sites, Cys-192 and Cys-193, have been identified. Furthermore, these adjacent Cys residues are cross-linked by a disulfide bond. In the quaternary structure of the receptor, the chains appear to be arranged in the order alpha gamma alpha beta delta around a central channel. Both the alpha and beta chains contribute to functionally significant NCI binding sites. The addition to receptor-rich membrane from Torpedo electric tissue of agonists (but not competitive antagonists) renders these NCI sites susceptible to photolabeling by the NCI quinacrine azide (QA). Furthermore, this susceptibility is transient, arising in milliseconds and subsiding in hundreds of milliseconds. These transiently susceptible sites are protected by other NCIs against photolabeling by QA. The time-course of the susceptibility and its dependence on agonist-concentration suggest that it might be the transient, rapid-onset desensitized state of the receptor that is most susceptible to photolabeling by QA.

The effect of Ca ion concentration on cross-bridge kinetics in a small bundle (one to three fibers) of chemically skinned rabbit psoas muscle is studied. The length of the muscle is oscillated in small amplitude sine waves (0.2% L0 peak-to-peak) at varying frequencies (0.125 -- 167 Hz), and the resulting amplitude and phase shift in tension are measured. The frequency response function (complex stiffness) thus obtained can be divided into three parts, which we name process (A) (centered at 1 Hz), process (B) (3--17 Hz), and process (C) (50 Hz). Process (B), which represents oscillatory work, further splits into two processes (B' and B) at partial Ca activation (less than 50% P0), where the phase-frequency plot appears W-shaped. The slower of the two processes (B') disappears by full activation, at which time the plot appears V-shaped. The characteristic frequencies associated with the minima of the plot do not shift in a graded way with Ca concentration, indicating that there is no change in apparent rate constants. Apparent rate constants of processes (A) and (C) are minimally affected by Ca. The above results are not altered when ionic strength is changed between 128 and 265 mM. We propose that activated thin filaments can have two "on" states and that Ca concentration controls the distribution of these two states. This mechanism generally supports the "switch" hypothesis of Ca regulation.