Calcium influx mediates the voltage-dependence of sperm entry into sea urchin eggs.

Sperm entry was monitored in voltage-clamped sea urchin eggs following insemination in a variety of artificial seawaters. In regular seawater, maintaining the membrane potential at increasingly negative values progressively inhibits sperm entry. Reducing [Ca(2+)](o) relieves the inhibition, shifting the sperm entry vs voltage relationship toward more negative potentials. Raising [Ca(2+)](o) shifts the relationship in the other direction. Large changes in [Na(+)](o) or [Mg(2+)](o) do not affect sperm entry although changing [Na(+)](o) dramatically changes the currents following sperm attachment. Applying one of seven different calcium channel blockers or replacing Ca(2+) with Ba(2+) or Sr(2+) or microinjecting calcium chelators into the cytoplasm relieves the block to sperm entry at negative potentials. We conclude that the block to sperm entry at negative potentials is mediated by calcium which crosses the membrane and acts at an intracellular site.

Measuring nerve excitation with polarized light.

There are changes in nerve birefringence and optical activity associated with nerve impulses. The birefringence response resembles the time integral of the gating current. Its magnitude suggests several hundred peptide bonds per channel reorient during excitation. The optical activity change has a different time course from the birefringence signal. Its amplitude is approximately consistent with the reorientation of alpha-helices within sodium channel molecules.

Chloramine-T alters the nerve membrane birefringence response.

The change in birefringence during depolarizing voltage-clamp pulses of internally perfused squid giant axons are biphasic. There is a rapid decrease in birefringence with a 220-microsec half time at 8 degrees C followed by a slow decrease over the next several milliseconds. After the pulse there is a rapid recovery which is smaller than the initial rapid decrease followed by a slow recovery phase. The rate of change of the slow phase during the pulse is more rapid for larger depolarizations. After the pulse the rate of change is more rapid for more negative potentials. 3.6 mM chloramine-T, applied externally until the sodium currents were prolonged and inactivation was removed, removed the slow phase of the birefringence response both during and after the pulse and made the fast 'off' response as large as the fast 'on' response. Two anesthetics reduced the birefringence response by about 20%. A rocking helix model is presented which relates the birefringence findings and earlier gating current experiments.

D2O and the sodium pump in squid nerve membrane.

In 10 K artificial seawater (ASW), D2O replacement reduced the Na efflux of squid axons by about one third. In 0 K ASW, D2O replacement had little effect. D2O reduced the K+ sensitivity of the efflux but increased the affinity for K+. A 4 degrees decrease in temperature mimicked the effects of D2O. When axons were injected with arginine, to decrease the ATP/ADP ratio, they lost K+ sensitivity in normal ASW, as expected. Their efflux into 0 K ASW became D2O sensitive. The results are discussed in terms of conformational changes in the Na pump molecular complex.

Molecular motion underlying activation and inactivation of sodium channels in squid giant axons.

Measurements of the changes in birefringence associated with changes in membrane potential were made with internally perfused squid giant axons in low sodium solutions at 0-8 degrees C. The time course of the birefringence changes share many properties of the 'gating' (polarization) currents previously studied in this nerve. Both can be demonstrated as an asymmetry in the response to voltage pulses symmetrical about the resting potential which is not present about a hyperpolarized holding potential. Both have a rapid relaxation, which precedes the sodium permeability change. Both exhibit an initial delay or rising phase. Both are reversibly blocked by perfusion with 30 mM or 300 nM tetrodotoxin. The birefringence response has a decrease in the amplitude of the rapid relaxation associated with the appearance of a slow relaxation. This is similar to the immobilization of fast gating charges which parallels sodium current inactivation. The amplitude of the birefringence and the gating current responses is consistent with a change in the alignment of several hundred peptide bonds per sodium channel.

Colchicine alters the nerve birefringence response.

The internal perfusion of squid axons with colchicine reversibly and selectively reduces the transient sodium current and the birefringence response to a brief depolarizing voltage pulse.

Effects of internal and external sodium on the sodium current-voltage relationship in the Squid giant axon.

The early transient current-voltage relationship was measured in internally perfused voltage clamped squid giant axons with various concentrations of sodium on the two sides of the membrane. In the absence of sodium on either side there is an outward transient current which is blocked by tetrodotoxin and varies with internal potassium concentration. The current increases linearly with voltage for positive potentials. Adding sodium ions internally increases the slope of the current-voltage relationship. Adding sodium ions externally also increases the slope between +10 and +80 mV. Adding sodium to both sides produces the sum of the two effects. The current-voltage relationships were fit by straight lines between +10 and +80 mV. Plotting the extrapolated intercepts with the current axis against the differences in sodium concentrations gave a straight line, Io = -P (Co-Ci)F. P, the Fickian permeability, is about 10(-4) cm/sec. Plotting the slopes in three dimensions against the two sodium concentrations gave a plane g = go + (aNao + bNai)F. a is about 10(-6) cm/mV-sec and b about 3 x 10(-6) cm/mV-sec. Thus the current-voltage relationship for the sodium current is well described by I = -P(Co-Ci)F+ (aco + bci)FV for positive potentials. This is the linear sum of Fick's Law and Ohm's Law. P/(a + b) = 25 +/- 1 mV (N = 6) and did not vary with the absolute magnitude of the currents. Within experimental error this is equal to kT/e or RT/F. Increasing temperature increased P, a and b proportionately. Adding external calcium, lithium, or Tris selectively decreased P and a without changing b. In the absence of sodium, altering internal and external potassium while observing the early transient currents suggests this channel is more asymmetric in its response to potassium than to sodium.

Sodium efflux from voltage clamped squid giant axons.

1. The efflux of radioactive sodium was measured from squid axons during simultaneous voltage clamp experiments such that it was possible to determine the efflux of sodium associated with a measured voltage clamp current. 2. The extra efflux of sodium associated with voltage clamp pulses increased linearly with the magnitude of the depolarization above 40 mV. A 100 mV pulse of sufficient duration to produce all of the sodium current increased the rate constant of efflux by about 10(-6). 3. Application of 100 nM tetrodotoxin eliminated the sodium current and the extra efflux of radioactive sodium. 4. Cooling the axon increased the extra efflux/voltage clamp pulse slightly with a Q10 of 1/1-1. On the same axons cooling increased the integral of the sodium current with a Q10 of 1/1-4. 5. Replacing external sodium with Tris, dextrose or Mg-mannitol reduced the extra efflux of sodium by about 50%. The inward sodium current was replaced with an outward current as expected. 6. Replacing external sodium with lithium also reduced the extra efflux by about 50% but the currents seen in lithium were slightly larger than those in sodium. 7. The effect of replacing external sodium was not voltage dependent. Cooling reduced the effect so that there was less reduction of efflux on switching to Tris ASW in the cold than in the warm. 8. The extra efflux of sodium into sodium-free ASW is approximately the same as the integral of the sodium current. Adding external sodium produces a deviation from the independence principle such that there is more exchange of sodium than predicted. Such a deviation from prediction was noted by Hodgkin & Huxley (1952c). 9. Using the equations of Hodgkin & Huxley (1952c) modified to include the deviation from independence reported in this paper and its temperature dependence, one can predict the temperature dependence of the sodium efflux associated with action potentials and obtain much better agreement than is possibly without these phenomena. 10. This deviation from independence in the sodium fluxes is the type expected from some kind of mixing and binding of sodium within the membrane phase.

The temperature dependence of the movement of potassium and chloride ions associated with nerve impulses.

1. The influx and efflux of radioactive potassium and chloride across the membrane of the squid giant axon were measured in resting and in stimulated nerves. The measurements were made at room temperature and at 6-8 degrees C. 2. At room temperature all eight flux measurements were comparable to previously reported values. 3. When the axons were cooled the resting potassium influx decreased with a Q10 of 1-9 and the resting potassium efflux decreased with a Q10 of 1-2. 4. With cooling the resting chloride efflux decreased with a Q10 of 1-3 and the resting chloride influx decreased with a Q10 of 2-8. This latter value, together with anomalous flux ratios for resting chloride fluxes may indicate an active uptake of chloride ions into the axon. 5. Cooling increased the extra efflux of potassium associated with nerve impulses with a Q10 of 1/1-5 and increased the extra influx of potassium with a Q10 of 1/3-3. 6. No extra efflux of chloride was detected at either temperature. Cooling produced no statistically significant change in the extra chloride influx but there was considerable scatter in the data. 7. Fluxes were computed as a function of temperature for standard action potentials with a variety of temperature coefficients for the conductances and rate constants. No single curve could match either the influx or the efflux data.

A comparison of radioactive thallium and potassium fluxes in the giant axon of the squid.

1. The influx and the efflux of 204Tl and 42K were measured in intact squid giant axons. 2. The resting efflux of 204Tl was found to be about one half of 42K and to have a temperature coefficient (Q10) of 1-3 as compared to 1-1 for K. 3. The extra efflux of 204Tl associated with nerve impulses was 30% greater than 42K. 4. From either Cl or NO3 sea water, the resting influx of 204Tl was about three times that of 42K. Ouabain reduced the influx of either isotope by about two thirds without changing the Tl/K ratio of the fluxes. This indicates that the Na pump can transport Tl. 5. From NO3 sea water the extra influx of 204Tl assoicated with nerve impulses was about the same as 42K. From Cl sea water there was no detectable extra influx of 204Tl. 6. The flux ratio, ouabain-insensitive influx/efflux, was different for the two ions. The resting flux ratio for Tl was consistent with a passive non-interacting flux, whereas K movements were consistent with 'single file' passage through the membrane. 7. The extra flux associated with nerve impulses is different from the resting flux both in Tl/K selectivity and in the effect of anion in the sea water. There is also a much higher flux per unit time during the nerve impulse. These differences suggest differences in the mechanisms underlying ion permeability at rest and during nervous activity.

The temperature dependence of the movement of sodium ions associated with nerve impulses.

1. The movement of sodium ions across the membrane of the squid giant axon was measured by the use of radioactive tracers. Unidirectional fluxes were measured at rest and when the nerve was stimulated. The difference was considered the extra flux association with nerve impulses.2. The extra influx in intact axons at room temperature was 5.5 p-mole/cm(2). impulse. At 6 degrees C the extra influx was 6.5 p-mole/cm(2). impulse giving a Q(10) of 1/1.2.3. In perfused axons a Q(10) of 1/1.6 was obtained for the extra sodium influx in bracketed experiments on individual axons.4. The Q(10) of the extra sodium efflux associated with nerve impulses was found to be 1/1.2 in intact axons.5. Hodgkin & Huxley had predicted a much larger temperature dependence for the extra fluxes. If this difference between prediction and experiment does not result from some experimental error, then the class of models for the ion fluxes suggested by Hodgkin & Huxley may be inapplicable.

Changes in light scattering that accompany the action potential in squid giant axons: potential-dependent components.

1. To obtain information about structural events that occur in axons, changes in light scattering from squid giant axons were measured during action potentials and voltage-clamp steps.2. The scattering changes were measured at several scattering angles. Because the changes in scattering divided by the resting scattering were between 10(-6) and 10(-5), signal-averaging techniques were used to increase the signal-to-noise ratio.3. The scattering changes during the action potential were different at different angles. Two types were found, one at 10-30 degrees (forward angles) and the other at 60-120 degrees (right angles).4. At forward angles, there was a transient scattering decrease during the action potential. The time course of the change was similar to that of the action potential; this change was thought to be potential-dependent.5. At right angles, there was a transient scattering increase during the action potential followed later by a second, longer-lasting increase. Indirect evidence indicated that neither component could be totally potential-dependent.6. To further analyse these effects, scattering was measured during voltage-clamp steps. The changes seen during hyperpolarizing steps were presumed to be potential-dependent; again two different changes were found, one at forward angles and one at right angles.7. The potential-dependent change at right angles occurred with a time course that could be approximated by a single exponential with a time constant tau = 24 musec. The change at forward angles required two exponentials, tau(1) = 23 musec, tau(2) = 900 musec, to represent its time course.8. The size of both potential-dependent changes was proportional to the square of potential. The change at right angles, but not that at forward angles, was increased in size by the addition of butanol or octanol to the bathing solution.

Changes in axon light scattering that accompany the action potential: current-dependent components.

1. When light scattering was measured during hyperpolarizing and depolarizing voltage-clamp steps, relatively large scattering changes were found during the depolarizing steps. These large changes were found to depend on the time integral of the ionic current and not on the changes in conductance or potential.2. The current-dependent changes were examined at several scattering angles, and three distinct time courses were found. At 30-120 degrees , the main change occurred after the current when steps of 2-5 msec duration were used. This change was called I-90 degrees . At 15-30 degrees , the change occurred with the same time course as the time integral of the current. This change was called I-25 degrees . At 5-15 degrees the scattering change occurred with a time course intermediate between that of I-90 degrees and I-25 degrees . This change was called I-10 degrees .3. In all experiments, outward potassium and outward sodium currents led to similar light scattering changes indicating that specific effects of the cation carrying the current across the membrane were not involved.4. The size of I-90 degrees was reduced by 29% when an isethionate artificial sea water was substituted for the normal chloride artificial sea water. This reduction equalled the reduction predicted for a transport number effect at the membrane-solution interface. The time course of I-90 degrees was similar to the predicted time course for a volume change in the periaxonal space, and such volume changes were tentatively identified as the origin of I-90 degrees .5. Because of difficulties in measuring the time course of I-25 degrees , it was not possible to distinguish between a water of hydration effect and a transport number effect as the cause of this change. Similarly, the origins of I-10 degrees were not identified. Only I-10 degrees was altered in size and time course when the external refractive index was increased with bovine albumin.6. When the scattering changes during the action potential were examined in light of the voltage-clamp experiments, we concluded that the forward-angle change was potential-dependent and that the long-lasting change at right angles probably represented a swelling of the periaxonal space resulting from the fact that chloride carried a significant fraction of the outward current during the action potential.