Voltage clamp experiments on internally perfused giant axons.

Abstract

METHODS MaterialThe experiments were done during the period November 1963 to January 1964. Giant axons of 585-870 it diameter were isolated from large specimens of Loligo forbei. Living squid were available occasionally but as a rule mantles which had been stored for a few hours in ice-cold sea water were used. Extrusion and perfusionThe uncleaned axons were extruded and perfused in the way described by Baker et al (1962a). The cell in which the axons were mounted and the method of changing internal solutions were also as described in that paper. The essential features of the cell, the electrodes and the electrical equipment are illustrated in Fig. 1. Description of internal electrodeThe internal electrode consisted of a 100 , glass capillary for measuring the internal potential and a 70g platinum wire, for sending current, attached to the capillary. The 100 t glass capillary was filled with 0-6 M-KCI in contact with a silver-silver chloride electrode. A 20 , bright platinum wire ran through the capillary to reduce the high frequency impedance. The shank was filled with an agar gel of 0-6 m-KCI to prevent bulk flow of KC1 out of the electrode. The external platinum wire was insulated except for the distal 10 mm which was left bare. This part, which extended for 5 mm on either side of the tip of the glass capillary, was carefully cleaned and platinized according to the method of Moore & Cole (1963). This reduced the impedance of the wire so that an increase in voltage corresponding to a change of about 40 Q) occurred from the beginning to the end of a 10 msec

Cation and anion sequences in dark-adapted Balanus photoreceptor.

“…If this is one of the well-known Eisenman sequences it can only be sequence X, while sequence IV was obtained for the resting state. Baker et al (1962) found sequence IV for the resting state squid axon and Chandler and Meves (1965) found that the active currents in squid axon were sequence XI. Further studies should help to establish other differe'nces between electrically excitable membranes and membranes that respond with graded potential changes.…”

Sodium and potassium conductance changes during a membrane action potential

“…For the experiments presented here the axons were perfused with potassium fluoride. It is known that potassium flows through the sodium channel with about 1/10 to 1/25 the ease of sodium ions (Chandler & Meves, 1965;Rojas & Atwater, Na AND K CONDUCTANCE CHANGES 1967). Thus for these measurements the situation for the sodium channels is equivalent to an internal sodium concentration which is some fraction of the existing potassium concentration.…”

“…This can be expressed in terms of conductances (see Fig. 6A): IC = GNa(VC -Na-ICRS) +GK (VC-VK -ICRs) + GL(VC-VL-ICRs), where 1Na, VK and VL are the reversal potentials for the Na+, and K+ and leakage channels respectively (Chandler & Meves, 1965 perfused Dosidicus fibres when Vc = Vi. The inward current transients do not all decline with the same rate constant as would be expected in the absence of a series resistance.…”

Voltage clamp experiments in striated muscle fibres

“…Apart from any injurious effect, the hypertonic solutions should reduce Vra by raising the internal concentrations of sodium and potassium. According to Chandler & Meves (1965) In order to obtain information about small sodium currents in the regenerative region, the method was modified by switching the feed-back control to the middle electrode, to improve stability, and increasing the electrode separations from 125 to 250 or 500 It, to increase sensitivity. In such experiments small graded inward currents were obtained up to a critical potential at which an all-or-nothing component appeared (Fig.…”