Summary. The effects of hydrostatic pressures up to 62 MPa upon the voltage-clamp currents of intact squid giant axons were measured using mineral oil as the pressure transmitting medium. The membrane resistance and capacitance were not appreciably affected over the whole range of pressures explored. The predominant effect of pressure is to slow the overall kinetics of the voltage-clamp currents 9 Both the early (Na) currents and the delayed (K) ones were slowed down by approximately the same time scale factor, which was in the range of 2 to 3 when pressure was increased from atmospheric to 62 MPa.Finer details of the effects, most evident at moderate depolarizations, are: the apparent initial delay in the turn-on of Na currents is increased by pressure less than is the phase of steepest time variation, and the later decay is slowed more than is the rising phase. The initial time course of the currents at high pressures can be made to overlap with that at normal pressure by a constant time compression factor, Ore, together with a small, voltage-dependent delay 9In a given axon, Om was fairly independent of voltage, and it increased exponentially with pressure according to an apparent activation volume, A V • ranging between 32 and 40 cmJ/mole. A V • tended to decrease with increasing temperature. Contrary to what is observed for moderate or large depolarizations, the kinetics of Na inactivation produced by conditioning prepulses of -50 or -60 mV was little affected over the whole range of pressures explored.Inferences about the pressure dependence of the steady-state Na activation were made from the comparison of the plots of early peak currents, Ip, versus membrane potential, E. The Na reversal potential, ENd, and the slope of the plots near EN, did not change significantly with pressure, but the peak Na conductance vs. E relationship was shifted by about + 9 mV upon increasing pressure to 62 MPa. Steady-state Na inactivation, h| was slightly affected by pressure. At 62 MPa the midpoint potential of the h~(E) curve, Eh, was shifted negatively by about 4 mV, while the slope at Eh decreased by about 38%.Under the tentative assumption that pressure directly affects the gating of Na channels, the Na activation data follows a simple Hodgkin-Huxley scheme if the opening of an m gate involves an activation volume of about 58 A_ 3 and a net volume increase of about 26 A 3. However, a self-consistent description of the totality of the effects of pressure on Na inactivation cannot be obtained within a similar simple context.

Voltage-clamp currents and resting membrane potential of squid giant axons have been studied at extracellular pH varying between 4 and 10. The membrane currents, analyzed according to the Hodgkin-Huxley equations, showed that sodium permeability, PNa(E), and potassium conductance gK(E), curves were shifted toward positive voltages by different amounts and slightly depressed as the external pH was lowered. Under the same conditions, taum(E) and taun(E) were found to be enhanced and shifted to a larger extent in the same direction. The rate constants alpham and alphan were shifted substantially toward positive voltages, but betam and betan changed hardly at all. The shift of the alpham(E) curve was analyzed in terms of a fixed surface charge model; it indicates that unspecific negative groups with an approximate pKa of 4.5 are located in the vicinity of sodium active sites with an average charge separation of 8 A. A similar figure is obtained for the potassium system from the shift of the alphan(E) curve.

Summary. The effect of pressure upon the delayed, K, voltageclamp currents of giant axons from the squid Loligo vulgaris was studied in axons treated with 300 nM TTX to block the early, Na, currents. The effect of TTX remained unaltered by pressure. The major change produced by pressures up to 62 MPa is a slowing down of the rising phase of the K currents by a time scaling factor which depends on pressure according to an apparent activation volume, AV*, of 31 cm3/mole at 15 ~ AV* increased to about 42 cm3/mole at 5 ~ Pressure slightly increased the magnitude, but did not produce any obvious major change in the voltage dependence, of the steadystate K conductance estimated from the current jump at the end of step depolarizations of small amplitude (to membrane potentials, E, < 20 mV) and relatively short duration. At higher depolarizations, pressure produced a more substantial increase of the late membrane conductance, associated with an apparent enhancement of a slow component of the K conductance which could not be described within the framework of the Hodgkin-Huxley (HH) n ~ kinetic scheme.The apparent A V* values that characterize the pressure dependence of the early component of the K conductance are very close to those that describe the effect of pressure on Na activation kinetics, and it is conceivable that they are related to activation volumes involved in the isomerization of the normal K channels. The enhancement of the slow component of membrane conductance by pressure implies either a large increase in the conductance of the ionic channels that are responsible for it or a strong relative hastening of their turn-on kinetics.

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The absorption of the lipophilic anions dipicrylamine (DPA-) and tetraphenylborate (TPhB-) by the lipid matrix of the squid axon membrane, and the kinetics of their translocation, were studied by the charge pulse relaxation technique. The axons were treated with tetrodotoxin (TTX) and 4-aminopyridine to block the ionic currents responsible for nerve excitation. At high enough concentrations of absorbed ions (approximately 10(-12) mol/cm2) the membrane voltage relaxation following a brief current pulse consisted mainly of two exponential components, whose time constants and relative amplitudes were used for estimating the translocation rate constant, K, and the density of absorbed ions, N. These measurements were performed at different hydrostatic pressures in the range 1-100 MPa (approximately 1,000 atm), and at different temperatures in the range 5 degrees C-20 degrees C. Both K and N were found to be little affected by pressure. The pressure dependence of K indicated that the translocation of lipophilic ions across the nerve membrane involves activation volumes of the order of 5 cm3/mol. In all experiments the passive membrane resistance was little affected by pressures up to 80 MPa. However, above 100 MPa it fell dramatically to low values, presumably because of phase separation phenomena between the membrane components. The temperature dependence of K, both for DPa- and TPhB-, implied an activation energy for ion translocation of the order of 60 kJ/mol, close to that measured in artificial lipid bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)