Nikolaus G. Greeff

Fractionation of the asymmetry current in the squid giant axon into inactivating and non-inactivating components

The operation of the voltage-sensitive sodium gating system in the nerve membrane involves conformational changes that are accompanied by small asymmetrical displacement currents. The asymmetry current may be divided into a component that is inactivated by positive voltage-clamp pulses, and recovers from inactivation with exactly the same time course as the sodium conductance, and one that is not inactivated. A method is described for recording the two components separately with the aid of an inactivating prepulse. They appear to have a marked difference in their rising phases, that of the non-inactivating component being just about as fast as the imposed step in membrane potential, while the inactivating component requires some tens of microseconds to reach its peak.

Kinetic analysis of the sodium gating current in the squid giant axon

A critical study has been made of the characteristics of the kinetic components of the sodium gating current in the squid giant axon, of which not less than five can be resolved. In addition to the principal fast component Ig2, there are two components of appreciable size that relax at an intermediate rate, Ig3a and Ig3b, Ig3a has a fast rise, and is present over the whole range of negative test potentials. Ig3babsent below -40 mV, exhibits a delayed onset and disappears on inactivation of the sodium system. There are also two smaller components, Ig1 and Ig4, with very fast and much slower relaxation time constants, respectively.

Long-term cultivation of cryopreserved human fetal brain cells in a chemically defined medium

Conditions for long-term cultivation of human fetal brain cells in a chemically defined medium were established using cryopreserved brain fragments obtained from legal abortions. Tissue of the same gestational age was pooled and the cells cultured in a fully defined medium containing insulin-like growth factors (IGF I and II). Primary cultures were kept for 2-4 weeks and secondary or tertiary cultures could be maintained for 3 months. The cultures were characterized by morphological, electrophysiological and biochemical methods. Glial cells were predominant during the first two weeks of culture. In later stages of cultivation, glial cells diminished in number and most cells were neuronal. Voltage-dependent Na+ channels were recorded from neurons. Biochemical studies indicated that the fetal brain cells contained and secreted immunoreactive somatostatin as well as the tachykinins, substance P and neurokinin A. Cultures grown in IGF II- or nerve growth factor-containing medium expressed increased choline acetyltransferase activity.

The Relationship between the Inactivating Fraction of the Asymmetry Current and Gating of the Sodium Channel in the Squid Giant Axon

A quantitative comparison between the voltage dependence of the inactivating component of the asymmetrical charge transfer in the squid giant axon and that of the sodium conductance indicates that activation of the sodium system involves either three subunits operating in parallel or a three-step series mechanism. This is confirmed by an examination of the relative timing of the flow of asymmetry and ionic currents during the opening and closing of the sodium channels. In agreement with previous suggestions, inactivation is coupled sequentially to activation. The evidence appears to argue against a trimeric system with three wholly independent subunits and favours a monomeric system that undergoes a complex sequence of conformational changes.

The quantal gating charge of sodium channel inactivation

Using a very low noise voltage clamp technique it has been possible to record from the squid giant axon a slow component of gating current (Ig) during the inactivation phase of the macroscopic sodium current (INa) which was hitherto buried in the baseline noise. In order to examine whether this slowIg contains gating charge that originates from transitions between the open (O) and the inactivated (I) states, which would indicate a true voltage dependence of inactivation, or whether other transitions contribute charge to slowIg, a new model independent analysis termed isochronic plot analysis has been developed. From a direct correlation ofIg and the time derivative of the sodium conductance dgNa/d the condition when only O-I transitions occur is detected. Then the ratio of the two signals is constant and a straight line appears in an isochronic plot ofIg vs. dgNa/d. Its slope does not depend on voltage or time and corresponds to the quantal gating charge of the O-I transition (qh) divided by the single channel ionic conductance (γ). This condition was found at voltages above − 10 mV up to + 40 mV and a figure of 1.21e− was obtained forqh at temperatures of 5 and 15°C. At lower voltages additional charge from other transitions, e.g. closed to open, is displaced during macroscopic inactivation. This means that conventional Eyring rate analysis of the inactivation time constant τh is only valid above − 10 mV and here the figure forqh was confirmed also from this analysis. It is further shown that most of the present controversies surrounding the voltage dependence of inactivation can be clarified. The validity of the isochronic plot analysis has been confirmed using simulated gating and ionic currents.

High resolution recording of asymmetry currents from the squid giant axon: Technical aspects of voltage clamp design

The design, realisation and performance of a voltage clamp system dedicated to recording asymmetry currents from the squid giant axon is presented. The design has been optimised with respect to dynamic response, signal-to-noise ratio and linearity. Analytical expressions are given for both the dynamic performance and noise characteristics which simplify the design and setting up of the clamp and provide excellent agreement between design theory and practice. A 0.5 cm2 area of membrane can be voltage clamped in response to a command input voltage step within 10 microseconds with smooth settling and up to 100% of the effective series resistance being compensated. The noise contributions of the recording chamber, voltage sensing electrodes and clamp electronics have been reduced such that recording of gating currents with a more than 10-fold reduction in the number of averages required compared with previous clamp designs is possible. The overall system nonlinearity results in typically less than 1% contribution to the measured asymmetry charge.

The early phase of sodium channel gating current in the squid giant axon

A fast component of displacement current which accompanies the sodium channel gating current has been recorded from the membrane of the giant axon of the squid Loligo forbesii. This component is characterized by relaxation time constants typically shorter than 25 µs. The charge displaced accounts for about 10% (or 2 nC/cm2) of the total displacement charge attributed to voltage-dependent sodium channels. Using a low noise, wide-band voltage clamp system and specially designed voltage step protocols we could demonstrate that this component: (i) is not a recording artifact; (ii) is kinetically independent from the sodium channel activation and inactivation processes; (iii) can account for a significant fraction of the initial amplitude of recorded displacement current and (iv) has a steady state charge transfer which saturates for membrane potentials above + 20 mV and below − 100 mV This component can be modelled as a single step transition using the Eyring-Boltzmann formalism with a quantal charge of 1 e− and an asymmetrical energy barrier. Furthermore, if it were associated with the squid sodium channel, our data would suggest one fast transition per channel. A possible role as a sodium channel activation trigger, which would still be consistent with kinetic independence, is discussed. Despite uncertainties about its origin, the property of kinetic independence allows subtraction of this component from the total displacement current to reveal a rising phase in the early time course of the remaining current. This will have to be taken into account when modelling the voltage-dependent sodium channel.

The Effect of Tetrodotoxin on the Sodium Gating Current in the Squid Giant Axon

The effect of tetrodotoxin (TTX) on the sodium gating current in the squid giant axon was examined by recording the current that flowed at the pulse potential at which the ionic current fell to zero, first in the absence and then in the presence of TTX. The addition of 1 μM TTX to the bathing solution had no consistent effect on the size of the initial peak of the gating current, but resulted in small changes in the timecourse of its subsequent relaxation which were mainly caused by a reduction of about one quarter in the component that has a delayed onset and may possibly arise from changes in the state of ionization of groups in the channel wall when the lumen fills with water. Our findings suggest that the binding of TTX at the outer face of the sodium channel does not interfere with the mechanisms of activation and inactivation by the voltage sensors, but has an allosteric effect on the access of internal cations to the inside of the channel.

Variable Ratio of Permeability to Gating Charge of rBIIA Sodium Channels and Sodium Influx in Xenopus Oocytes

Whole-cell gating current recording from rat brain IIA sodium channels in Xenopus oocytes was achieved using a high-expression system and a newly developed high-speed two-electrode voltage-clamp. The resulting ionic currents were increased by an order of magnitude. Surprisingly, the measured corresponding gating currents were approximately 5-10 times larger than expected from ionic permeability. This prompted us to minimize uncertainties about clamp asymmetries and to quantify the ratio of sodium permeability to gating charge, which initially would be expected to be constant for a homogeneous channel population. The systematic study, however, showed a 10- to 20-fold variation of this ratio in different experiments, and even in the same cell during an experiment. The ratio of P(Na)/Q was found to correlate with substantial changes observed for the sodium reversal potential. The data suggest that a cytoplasmic sodium load in Xenopus oocytes or the energy consumption required to regulate the increase in cytoplasmic sodium represents a condition where most of the expressed sodium channels keep their pore closed due to yet unknown mechanisms. In contrast, the movements of the voltage sensors remain undisturbed, producing gating current with normal properties.

Experimental evidence for saltatory propagation of the Mauthner axon impulse in the tench spinal cord

External longitudinal current recording was applied in situ to the exposed spinal cord of the tench (Tinca tinca) for the study of impulse propagation in the Mauthner axon, a giant nerve fibre whose myelin sheath is not interrupted by nodes of Ranvier. Impulses in the antidromically excited Mauthner axon were recorded from the dorsal surface of the spinal cord; the time-lag between the main peaks of the bipolarly recorded current signal and unipolarly recorded reference signal was measured at regular intervals along the cord. Using a slot width of the bipolar electrodes, d = 0.65 mm and electrode displacement s = 0.5 mm, the latency plotted as a function of distance showed small fluctuations, but no clear-cut steps over a 12.5 mm stretch of spinal cord. However, with improved spatial resolution (d = 0.24 mm, s = 0.1 mm) and electrical insulation of the spinal cord from the underlying tissues, it was possible to demonstrate steps in the latency plot occurring at 0.5--0.3 mm intervals and indicating a saltatory propagation of the Mauthner axon impulse. The distances between the latency steps and their distribution was comparable to the known distribution of the Mauthner axon collaterals suggesting that the myelin-free regions of the collaterals may be quivalents of Ranvier nodes.