7. All observations could be well simulated by a two-step model for inactivation in which myoplasmic free calcium equilibrates rapidly with a high-affinity calcium Authors' names appear in alphabetical order. * Present address and address for correspondence and reprint requests. 8. An alternative model in which calcium binds to a soluble receptor (e.g. free calmodulin) and then the calcium-receptor complex binds to and directly inactivates the channel was shown to be formally identical to the preceding model. Either model could closely simulate all observations.

Vagus nerve stimulation (VNS) is effective in refractory epilepsy and depression and is being investigated in heart failure, headache, gastric motility disorders and asthma. The first VNS device required surgical implantation of electrodes and a stimulator. Adverse events (AEs) are generally associated with implantation or continuous on−off stimulation. Infection is the most serious implantation‐associated AE. Bradycardia and asystole have also been described during implantation, as has vocal cord paresis, which can last up to 6 months and depends on surgical skill and experience. The most frequent stimulation‐associated AEs include voice alteration, paresthesia, cough, headache, dyspnea, pharyngitis and pain, which may require a decrease in stimulation strength or intermittent or permanent device deactivation. Newer non‐invasive VNS delivery systems do not require surgery and permit patient‐administered stimulation on demand. These non‐invasive VNS systems improve the safety and tolerability of VNS, making it more accessible and facilitating further investigations across a wider range of uses.

To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.

SUMMARY1. Intramembrane charge movement and myoplasmic free calcium transients (A[Ca2+]) were monitored in voltage-clamped segments of isolated frog muscle fibres cut at both ends and mounted in a double Vaseline-gap chamber. The fibres were stretched to sarcomere lengths of 3-54-6 ,tm to minimize mechanical movement and the related optical artifacts.2. The over-all calcium removal capability of each fibre was characterized by analysing the decay of A[Ca2+] following pulses of several different amplitudes and durations. The rate of sarcoplasmic reticulum (s.r.) calcium release was then calculated for each A[Ca2+] using the calcium removal properties determined for that fibre.3. The calculated calcium release wave form reached a relatively early peak and then declined appreciably during a 100-150 ms depolarizing pulse. The voltage dependence of the peak rate of calcium release was steeper and was centred at more positive membrane potentials than the steady-state voltage dependence of charge movement in the same fibres.4. A considerable fraction of the total intramembrane charge was moved at potentials at which A[Ca2+] and calcium release were only a few per cent of maximum.This 'subthreshold' charge may correspond to charge moved in preliminary transitions that precede a final charge transition that activates release. 5. A 'stepped on' pulse protocol was used to experimentally separate the subthreshold charge movement from the charge movement of the final transitions that may control calcium release. The stepped on pulse consisted of a set 50 ms pre-pulse to a potential just at or below the potential for detectable A W. MELZER AND OTHERS 6. For a wide range of test pulse amplitudes and durations in the stepped on protocol the peak rate of calcium release was linearly related to the charge movement during the test pulse. This result points to a tight control of activation of s.r. calcium release by intramembrane charge movement.7. The voltage dependence of both charge movement and of the rate of calcium release could be fitted simultaneously with a three-state, two-transition sequential model in which charge moves in both transitions but only the final transition activates s.r. calcium release. A model with three identical and independent charged gating particles per channel gave an equally good fit to the data. Both models closely fit the charge movement and release data except within about 10 mV of the voltage at which release became detectable, where release varied more steeply with membrane potential than predicted by either model. 8. Fits with the three-state model gave about equal amounts of charge movement in each transition but a 30-50 mV difference in the mid-point voltages for the two transitions. The single channel current calculated for the s.r. calcium channel based on this model was similar to values obtained for single surface membrane calcium channels in other preparations under similar transmembrane conditions.

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