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A comparison of propagated action potentials from tropical and temperate squid axons: different durations and conduction velocities correlate with ionic conductance levels

Joshua J. C. Rosenthal and Francisco Bezanilla^*

Departments of Physiology and Anesthesiology, UCLA School of Medicine, Los Angeles, CA 90095, USA

View larger version (19K): [in a new window]   Fig. 1. Propagated action potentials from the giant axons of four species of squid. Single propagated action potentials were recorded from giant axons using intracellular microelectrodes. (A) Action potentials from four species. (B) Action potentials from A superimposed by aligning peaks. All recordings were made at 15°C. Axons were bathed in 10K ASW (see Materials and methods).

View larger version (24K): [in a new window]   Fig. 2. Analysis of propagated action potentials. Measurements of propagated action potential rise time, fall time and conduction velocity in four species of squid. , Loligo opalescens; , L. pealei; , L. plei; , Sepioteuthis sepioidea. Values are means ± S.E.M. (A) Schematic diagram of how measurements were made. Both rise time and fall time were measured between 10 and 90% of the voltage difference between rest and action potential peak. Conduction velocities were taken from experiments using two electrodes, and velocities were measured by dividing the distance between the electrodes by the time between the two action potential peaks. (B) Graph of rise time versus temperature. The ordinate is plotted on a logarithmic scale. L. opalescens, N=6; L. pealei, N=18; L. plei, N=9; S. sepioidea, N=9. (C) Graph of fall time versus temperature. The ordinate is plotted on a logarithmic scale. L. opalescens, N=6; L. pealei, N=19; L. plei, N=9; S. sepioidea, N=9. (D) Graph of conduction velocity versus temperature. Conduction velocities were normalized to the square root of the axon's diameter. L. opalescens, N=4; L. pealei, N=11; L. plei, N=5; S. sepioidea, N=6. (E) Ratio of fall time to rise time versus temperature (data from B and C).

View larger version (15K): [in a new window]   Fig. 3. Separation of Na+ and K+ currents from voltage-clamped axons. (A) Voltage-clamp recording from a Sepioteuthis sepioidea axon at 20°C. The arrow indicates the start of a voltage step to 0 mV. (B) Family of K+ currents from a S. sepioidea axon at 20°C in the presence of tetrodotoxin (TTX). At the arrow, voltages were stepped from the holding potential to -40 mV, -20 mV, 0 mV, 20 mV, 40 mV and 60 mV. (C) Measurement of Na+ current (INa) in a Loligo plei axon at 10.6°C by TTX subtraction. (i) Two traces after voltages had been stepped from the holding potential to -10 mV, before and after TTX, superimposed. (ii) INa resulting from the TTX trace being subtracted from non-TTX trace. (iii) A family of INa traces from the same axon, using the same technique, after voltage steps to -30 mV, -10 mV, 10 mV, 30 mV, 50 mV and 70 mV. All holding potentials were -70 mV. The external solution was 10K ASW (see Materials and methods). The first points of each trace are at 0 µA.

View larger version (23K): [in a new window]   Fig. 4. Analysis of Na+ conductance (gNa) and K+ conductance (gK) from voltage-clamped axons. All analyses are derived from experiments at 10°C. , L. pealei; , L. plei; , Sepioteuthis sepioidea. Values are means ± S.E.M. (A) gNa, normalized to membrane surface area, versus voltage. L. pealei, N=5; L. plei, N=5; S. sepioidea, N=9. Values for S. sepioidea are significantly different (P0.05) from those for either species of Loligo between O mV and +30 mV (at +40 mV, P=0.06). (B) gK, normalized to membrane surface area, versus voltage. L. pealei, N=5; L. plei, N=7; S. sepioidea, N=8. Values are significantly different between L. plei and L. pealei at all voltages +20 mV and between L. plei and S. sepioidea at all voltages 0 mV. (C) gNa, normalized to maximum values of gNa, versus voltage. (D) gK, normalized to maximum values of gK, versus voltage. (E) Half-time (t1/2) for INa versus voltage. (F) t1/2 for IK versus voltage. t1/2 was defined as the time after the voltage step when half the maximum current was activated. Vm, membrane potential.

View larger version (16K): [in a new window]   Fig. 5. Measurement of the reversal potential during a membrane action potential. All recordings were taken from a Loligo pealei axon at 15°C. Holding potential, just before current clamping, was -65 mV. Axons were bathed in 10K ASW (see Materials and methods). (A) A single membrane action potential. (Bi) Seven similar action potential recording, which have been voltage-clamped after 700 µs, superimposed. Voltage-clamp potentials are from -70 mV to +50 mV in increments of 20 mV. (Bii) Paired current traces for voltage traces in Bi demonstrating instantaneous jumps. (C) Results from nine experiments similar to that in B but interrupted at different times. Reversal potential (Erev), determined from instantaneous current/voltage plots, plotted as a function of time and superimposed onto an action potential.

View larger version (24K): [in a new window]   Fig. 6 . Measurement of the K+ conductance (gK) and Na+ conductance (gNa) during a membrane action potential. All recordings were made at 10°C. Holding potential was -65 mV. Axons were bathed in 10K ASW (see Materials and methods). For gK experiments, time points are at 100 µs intervals for Loligo pealei and at 200 µs intervals for L. plei and Sepioteuthis sepioidea. For gNa, time points are at 20 µs intervals for L. pealei and at 50 µs intervals for L. plei and S. sepioidea. (Ai) Membrane action potential from L. pealei interrupted by voltage-clamp to -70mV (K+ equilibrium potential, EK) after 700 µs (arrow). (Aii) Matching current recording. The value of the instantaneous current at the time of voltage-clamp switch-on corresponds to the Na+ current (INa). (Bi) Membrane action potential from L. pealei interrupted by voltage-clamp to +55 mV (Na+ equilibrium potential, ENa) after 2 ms (arrow). (Bii) Matching current recording. The value of the instantaneous current at the time of voltage-clamp switch-on corresponds to the K+ current (IK). (C—E) Experiments similar to those in A and B, performed at many `interruption' points were used to derive time courses for gK and gNa from axons of L. pealei, L. plei and S. sepioidea. In each panel, an action potential (solid line) is superimposed on the time course for gNa ([UNK]) and gK ().

View larger version (39K): [in a new window]   Fig. 7 . Analysis of the K+ conductance (gK) and Na+ conductance (gNa) during a membrane action potential. All recordings were made at 10°C. The holding potential before current-clamp was -65 mV. Axons were bathed in 10K ASW (see Materials and methods). , L. pealei; , L. plei; , Sepioteuthis sepioidea. Values are means ± S.E.M. For all data, the action potential peak is considered to be time zero, and all time measurements are shifted accordingly. For gK experiments, data points are at 50 µs intervals for L. pealei and at 200 µs intervals for L. plei and S. sepioidea. For gNa, time points are at 20 µs intervals for L. pealei and at 50 µs intervals for L. plei and S. sepioidea. (A) Time course of gNa (per surface area) versus time. N=5 for all species. (B) gNa normalized to peak values versus time (same data as in A). (C) Time course of gK (per surface area) versus time. N=4 for all species. (D) gK normalized to peak values versus time (same data as in C).