Lobster Stretch Receptor Neurone Research Articles

The K–Cl Cotransporter in the Lobster Stretch Receptor Neurone—A Kinetic Analysis

Experiments were performed to define quantitatively the substrate (K + and Cl −) dependence of the transport function (production of equally large and oppositely directed K +and Cl − flows/currents) of an earlier (Theander et al., 1999) identified electroneutral K–Cl cotransporter in the slowly adapting stretch receptor neurone of the European lobster. The experiments were based on microelectrode techniques. This allowed us to perform steady-state measurements of the so-called “instantaneous” current–voltage relationships (around a holding voltage of −65 mV after a blockage of the cell's action potential and hyperpolarization-activated currents) and intracellular ion concentrations at various settings of the extracellular K + and Cl − concentrations. From the results, we could then define steady-state values of all of the cell's non-KCl cotransporter K + and Cl − currents. Finally, the negative sums of the inferred non-KCl cotransporter K + and Cl − currents could be taken as equivalents of the K–Cl cotransporter's K + and Cl − currents for the reason that, in steady state, all membrane currents add up to zero. For the cotransporter currents, thus inferred for a range from 2.5/410.5 to 40.0/448.0 mM external K +/Cl −, we found that their absolute values increased in a nonlinear fashion from about 5 nA cell −1 at the lowest, to about 20 nA cell −1 at the highest external K +/Cl − concentrations. Formally, this relationship could be reproduced by a Hill function-based enzyme kinetic expression simulating inward and outward transmembrane electroneutral ion transports. Following insertion of this expression into a comprehensive model of electrical membrane functions and intracellular solute and solvent control in the lobster stretch receptor neurone, the model predictions suggested that the K–Cl cotransporter does play an important role in (a) keeping intracellular Cl − low for a proper function of the cell's inhibitory system, and (b) enabling rapid transmembrane K + shifts that provide for a stabilization of the cell's membrane voltage and membrane excitability in cases of varying extracellular K + concentrations. The model predictions gave, however, no clear evidence that the K–Cl cotransporter is critically involved in the cell's volume regulation in conditions of varying extracellular osmolalities.

Cl- transport in the lobster stretch receptor neurone.

Experiments were performed to identify mechanisms underlying non-leakage and non-H+/HCO3--linked transmembrane Cl- transports in the slowly adapting stretch receptor neurone of the European lobster, using intracellular microelectrode and pharmacological techniques. In methodological tests, it was established that direct estimates of intracellular Cl- with ion-sensitive microelectrodes are statistically identical with indirect estimates by means of a GABA method, where 1-2 mM GABA is transforming the cell's membrane voltage into its Cl- equilibrium voltage from which the Cl- concentration is inferred by the Nernst equation. From experiments using sodium orthovanadate and ethacrynic acid, supposed to block primary Cl- pumps, and bumetanide, supposed to block Na-K-Cl co-transporters, it appeared that neither of the two Cl- transport systems exists in the stretch receptor neurone. It could be shown, however, that the cell is equipped with an electroneutral K-Cl co-transporter that (a) is blockable by furosemide in high (Km approximately 350 microM), by 4-acetamido-4'-isothiocyanato-stilbene-2,2-disulphonic acid (SITS) in medium-high (Km approximately 35 microM), and by 4, 4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) in low (Km approximately 15 microM) doses, (b) is (transiently) activatable by (1 mM) n-ethylmaleimide, (c) is not suppressed by extracellular Rb+ or NH4+, and (d) is not directly coupled to any transmembrane transports of Na+, H+ or HCO3-. From functional tests, with varying transmembrane K+ and Cl- gradients, evidence obtained that the K-Cl co-transporter is able to reverse its transport direction and to adjust its transport rate in a considerable range. As a whole, the results speak in favour of the K-Cl co-transporter being responsible (a) for normally keeping the intracellular Cl- concentration at low levels, for an optimization of the cell's inhibitory system, and (b) for achieving fast transmembrane shifts of K+ (and Cl-), as a means of stabilizing the cell's membrane excitability in conditions of varying extracellular K+ concentrations.

Properties of an A-current in slowly and rapidly adapting stretch receptor neurones of lobster

A previously unnoticed outward membrane current has been identified, characterized and specified as a so-called A-current in the slowly and rapidly adapting lobster stretch receptor neurone. In both cells the current was, after blockage of a tetrodotoxin-sensitive Na + current and a tetraethylammonium- and 4-aminopyridine-sensitive delayed rectifier current, seen to activate fully within about 25 ms of square-shaped depolarizations beyond voltage levels of -40 to -30 mV and, then, to inactivate completely with a (voltage independent, within the voltage span under observation) time constant of 110 ms. The A-currents of rapidly and slowly adapting receptors were noticed to differ significantly from each other in that the A-current of the rapidly adapting cell is activated, and inactivated, at 10-15 mV more negative voltage levels than the A-current of the slowly adapting cell. Also, the maximum permeability of the A-channel system appeared to be distinctly larger in the rapidly than in the slowly adapting cell. Both of these circumstances were able to explain why, at a given level of membrane depolarization, a markedly stronger A-current is activated in the rapidly than in the slowly adapting cell. On the basis of experimental data it was possible to formulate a mathematical A-current description which was incorporated into a previously published model of the lobster stretch receptor neurone. Using this model, evidence was obtained that the A-current may play a functionally significant role (in the rapidly adapting cell) by increasing the speed of action potential repolarization and thereby enhancing the cell's dynamic stimulus sensitivity.

Analysis of leak current properties in the lobster stretch receptor neurone.

Experiments were performed to characterize the so-called leak current of the slowly adapting stretch receptor neurone of the European lobster with respect to its ionic basis, its kinetics and its pharmacology. Estimates of the leak current were obtained by subtraction of a Na-K pump current and of an unspecific impalement current from a non-dynamic ('instantaneous') current, recorded in a voltage range from approximately -120 to approximately -30 mV, after blockage of spike-generating currents and a hyperpolarization-activated inwardly rectifying current (Q-current). The leak current, estimated in this way, was seen to reverse direction at the cell's K+ equilibrium voltage, thus indicating that it is carried by K+ passing through channels which, also, proved to be permeable to Rb+ and NH4+, but not permeable to Na+ or Cl- to any significant extent. Kinetically, the leak current was found to be characterized by being enhanced by increases in extracellular K+ and by being subject to outward rectification, most distinctly at elevated extracellular [K+]. In quantitative terms, these kinetic properties could be accounted for by a mathematical model comprising (1) a one-site two-barrier Eyring formulation describing ion permeation through membrane channels and (2) an ordinary dose-response relationship describing the channel-opening effect of K+ at an extracellular regulatory site. Pharmacologically, the leak current proved to be distinguished by being reversibly blockable, in a non-voltage dependent manner, by CO2+ (Kd = 0.9 mM, Hill coefficient 1.1) and procaine, but not by Ba2+, Gd3+, bupivacaine (a local anesthetic), or other K+ channel blockers such as TEA, 4-AP and Cs+. It is concluded that, in native unimpaled cells, the K+ carried leak current (1) is setting the resting voltage together with the (mainly) Na(+)-carried Q-current and the Na-K pump current, (2) is determining the cell's firing threshold, together with the spike generating currents, and (3) is also stabilizing the cell's membrane excitability in conditions of varying extracellular [K+], by virtue of its K+ sensitivity.

Functional effects of a hyperpolarization-activated membrane current in the lobster stretch receptor neurone.

The functional effects of a hyperpolarization-activated membrane current (IQ) in the slowly adapting lobster stretch receptor neurone were investigated. From comparisons between changes in membrane excitability due to blockage of IQ by Cs+, in normally impaled and native unimpaled (Edman et al. 1987 b) cells, it could be concluded that the resting voltage of native cells is distinctly more negative than -65 mV (average membrane voltage of impaled cells) and, therefore, under the control of an activated IQ. Starting from this conclusion, impaled cells were polarized to holding (resting) voltages around -75 mV and their polarization and excitability properties studied after tetanic impulse activity and variation of various external influences (K+, pH, temperature), both in control conditions and after blockage of IQ by 2 mM Cs+. It was found that an unblocked IQ (a) greatly accelerates the initial (90%) decay of post-tetanic hyperpolarization, and (b) depresses distinctly any polarization and excitability alterations due to increases in extracellular K+ concentration (from 2.5 to 10 mM), variations in extracellular pH (between 6.4 and 8.6), and changes in temperature (between 14 and 24 degrees C). It was inferred that in well polarized cells, IQ plays a role as a stabilizer of membrane polarization and excitability in conditions of varying external influences. From a model study of IQ it could be concluded that, with its slow dynamic responses, the current is well adapted to its functional purposes and to the rather slow homeostatic effects of the cell's Na-K pump.

Ion (H+, Ca2+, Co2+) and temperature effects on a hyperpolarization‐activated membrane current in the lobster stretch rceptor neurone

The effects of some ions (H+, Ca2+, Co2+) and temperature on the Q-channel (a channel which conducts a hyperpolarization-activated membrane current, IQ) was investigated in the slowly adapting lobster stretch receptor neurone. It was found: (1) that increases in pH cause an increase in the maximum channel conductance; (2) that Co2+ (1 mM) and increases in extracellular Ca2+ produce a positive shift of the relationship between the membrane voltage and the steady-state channel activation along the voltage scale; (3) that Co2+ and decreases in extracellular pH cause a decrease in rate of the voltage-dependent step of the channel's gating reaction; (4) that none of the ions has an effect on the rate of the voltage-independent step of the gating reaction; and (5) that Co2+ and K+ do not interfere with each other's actions on the channel's gating reaction and maximum ion conductivity. Increases in temperature from 13 to 23 degrees C were found: (1) to cause an increase in the maximum Q-channel conductance by a factor of 1.7; (2) to have no effect on the voltage dependence of the channel's gating reaction; but (3) to lead to an increase in rate of the gating reaction by a factor of about five for the voltage-dependent, and by a factor of about three for the voltage-independent gating step. The temperature sensitivity of the IQ mechanism proved to be mathematically reproducible by a previously developed, and slightly modified Q-channel model (Edman et al. 1987, Edman & Grampp 1989), and to be of no significance in the control of membrane excitability in a thermally unstable environment.

Ion permeation through hyperpolarization-activated membrane channels (Q-channels) in the lobster stretch receptor neurone

In the lobster stretch receptor neurone it is possible to demonstrate a hyperpolarization-activated membrane current, IQ, which appears to be carried by Na+ and K+ in combination. The ion permeability of the membrane channel conducting this current (Q-channel) was investigated using conventional electrophysiological techniques including intracellular ion concentration measurements. It was found that none of the ions choline, protonated Tris, Rb+, NH4+, Li+, and protonated hydroxylamine was able to pass through the Q-channel which, thus, appears to be permeable to Na+ and K+ only. With increasing extracellular Na+ concentrations, IQ was increased up to a saturation level. This behaviour could be described by a one-site-two-barriers version of the Eyring rate theory, assuming that the permeant ions are turned over at specific saturable channel sites which 'sense' 70% of the transmembrane potential difference. With increasing extracellular K+ concentrations, IQ was increased in accordance with a simple first-order dose-response relationship. This finding can be accounted for by assuming that K+ increases all rates of turn-over of the permeant ions at their specific sites by similar relative amounts. Changes in extracellular Na+ and K+ concentrations were found to have no effect on the gating properties of the Q-channel.

Analysis of gated membrane currents and mechanisms of firing control in the rapidly adapting lobster stretch receptor neurone.

1. The gated membrane currents (a tetrodotoxin-sensitive Na+ current and a tetraethylammonium- and 4-aminopyridine-sensitive K+ current) of the rapidly adapting stretch receptor neurone of lobster were investigated with respect to their kinetic properties using electrophysiological, pharmacological, and mathematical techniques. 2. The currents were found to be controlled by slow inactivations as well as by fast Hodgkin-Huxley (1952) gating processes. They could be described by kinetic expressions which differed from those inferred for the slowly adapting receptor (Gestrelius & Grampp, 1983a; Gestrelius, Grampp & Sjölin, 1983) only with respect to some of the parameter values. 3. With these expressions, and additional equations for the cell's pump and leak current components (Edman, Gestrelius & Grampp, 1986), a mathematical receptor model was formulated which accurately predicts the impulse activity of the living preparation in different functional circumstances and which, therefore, was adopted as an appropriate theory of firing regulation. 4. From a model analysis it appeared (a) that the 'rapid' adaptation of the receptor's impulse activity is mainly an effect of slow Na+ current inactivation starting a regenerative process of accommodation which, basically, is due to a small ratio of subthreshold Na+ to K+ currents; (b) that, because of the transmembrane Na+ influx being limited by accommodation, impulse firing is only little affected by a Na+-dependent pump current activation; and (c) that the phenomenon of increased firing frequency initially during prolonged stimulation ('negative adaptation') is an effect of the slow K+ current inactivation being faster than the slow Na+ current inactivation at comparable degrees of membrane polarization. 5. From further model studies it also appeared that, during depolarizations between successive action potentials evoked by constant stimulation, the membrane behaves like a high-resistance constant-current generator feeding into a short-circuiting capacitor. In consequence, the cell's stimulus sensitivity (change in firing frequency with stimulation strength) is, at functionally relevant stimulation intensities, mainly determined by the membrane capacitance and by the amplitude of the interspike membrane depolarization while, at higher stimulation intensities and firing frequencies, it becomes more and more a function of the spike duration itself.

Current activation by membrane hyperpolarization in the slowly adapting lobster stretch receptor neurone.

1. A polarization-induced membrane current, IQ, was investigated in the slowly adapting lobster stretch receptor neurone using conventional electrophysiological techniques including intracellular ion measurements. 2. The current was readily blocked by Cs+ in a voltage-dependent manner, but proved to be unaffected by tetrodotoxin, tetraethylammonium and 4-aminopyridine. 3. From an analysis of the ionic basis of IQ, it appeared that the current is carried by both Na+ and K+ through a membrane channel whose permeability for K+ is about six times larger than that for Na+ in a normal ionic environment. In the presence of reduced external Na+ concentration the Q-channel increases its permeability for both Na+ and K+, but more so for Na+ than for K+. 4. Kinetically, IQ was found to be characterized by a steep sigmoidal relationship between membrane voltage and steady-state current activation, and by a bell-shaped relationship between membrane voltage and the time constant of the exponential phase of current activation or deactivation. A significant feature of the latter relationship is a tendency to level off at finite time-constant values in both strongly hyperpolarizing and strongly depolarizing voltage regions. 5. From the experiments a mathematical IQ model was inferred. This model was based on constant-field and channel-gating kinetics involving a voltage-dependent reaction step in series with a voltage-independent reaction step. The model was found to successfully reproduce IQ behaviour in the living preparation. 6. Functionally, the activation of IQ was found to play a role in setting the cell's resting polarization and membrane excitability. This function was inferred from experiments on unimpaled cells in which it was possible to demonstrate some overlap between the voltage ranges of IQ activation and impulse initiation. In addition, in impaled cells the activation of IQ was found to cause some shortening of post-tetanic membrane hyperpolarization and to accelerate, thereby, the post-tetanic restoration of membrane excitability to control levels.

Impulse firing in the slowly adapting stretch receptor neurone of lobster and its numerical simulation.

A mathematical model of the electrical activity of the slowly adapting lobster stretch receptor neurone is presented. The model is based on constant field and state transition theory and employs measurements of the kinetics of membrane currents in sub- and near-threshold voltage regions (Gestrelius, Grammp & Sjölin 1981, Gestrelius & Grampp 1983, Gestrelius, Grampp & Sjölin 1983, Edman, Gestrelius & Grampp 1983). In addition to the classical action potential generating mechanisms (Hodgkin & Huxley 1952) the model also includes the processes of slow Na and K inactivation, ion flux dependent changes of the intracellular Na+ and K+ concentrations, and the activity of an electrogenic Na-K pump sensitive to intracellular Na+ accumulation. The model is able to correctly simulate recorded action potentials as well as repetitive firing both with respect to stimulus dependence (sensitivity) and time dependence (adaptation) during prolonged electrical stimulation. In the living cell firing adaptation is found to consist of an initial phase with a relatively high, and a later phase with a lower rate of adaptation. From the model properties it can be concluded that the initial phase is mainly caused by the slow Na inactivation, while the later phase is due to a slow Na+ influx dependent pump current activation.

Intracellular ion control in lobster stretch receptor neurone.

The control of intracellular ion concentrations by means of passive and active transmembrane ion transports was investigated in the lobster stretch neurone using electrophysiological and pharmacological techniques in combination with recording with ion-sensitive microelectrodes. In resting conditions [Na+]i, [K+]i, and [Cl-]i were, in both slowly and rapidly adapting cells, found to be in the order of 20, 155, and 50 mM, respectively. In the slowly adapting cell impulse firing at stationary frequencies of 7-10 Hz caused an increase in [Na+]i and a decrease in [K+]i of 20-30 mM; [Cl-]i was only little affected, the rise in [Na+]i led to an enhanced Na-K pump activity noticeable as an increase in pump current production. In stationary conditions the quotient between pump current and Na+ influx increments was about 0.3, which is compatible with 3:2 Na-K pumping ratio in the present preparation. From measurements of the pump current activation during stationary firing at maximum tolerable frequencies an estimate was made of the cell's maximum pump current production. The measurements were used in the formulation of a mathematical model of the intracellular ion control in which expressions of active and passive transmembrane ion transports are incorporated into the continuity equation for the ion fluxes involved.

Kinetics of the TTX sensitive Na+ current in the slowly adapting lobster stretch receptor neurone.

The kinetics of the TTX sensitive Na+ current (INa) in the slowly adapting lobster stretch receptor neurone were investigated in sub- and near-threshold voltage regions using electrophysiological and pharmacological techniques. In dynamic conditions INa was found to display both fast and slow reactions. These were attributed to a fast Hodgkin-Huxley type of Na activation and inactivation, and a slow type of Na inactivation, respectively. In stationary conditions the voltage dependence of the slow Na inactivation was shifted in a depolarizing direction by increasing, and in a hyperpolarizing direction by decreasing the extracellular Ca++ concentration. From this finding as well as from its kinetic properties the slow Na inactivation was classified as a genuine gating process. The processes of fast Na activation and inactivation were too fast for a dynamic analysis with the recording technique available. An estimate of their stationary voltage dependence could however be obtained in a voltage range from about -80 to about -50 mV. The experimental findings were used for the formulation of a mathematical description of INa in the present preparation based on constant field and state transition theories.

Kinetics of the TEA and 4-AP sensitive K+ current in the slowly adapting lobster stretch receptor neurone.

The kinetics of the TEA and 4-AP sensitive K+ current (IK) in the slowly adapting lobster stretch receptor neurone were investigated in sub- and near-threshold voltage regions using electrophysiological and pharmacological techniques. In dynamic conditions IK was found to display both fast and slow reactions. These were attributed to a Hodgkin-Huxley type of K activation, and a slow type of K inactivation, respectively. The slow K inactivation could be shown to be unrelated to K+ flux dependent changes in intra- and pericellular K+ concentrations. Its stationary voltage dependence was however shifted in a depolarizing direction by increasing, and in hyperpolarizing direction by decreasing the extracellular Ca++ concentration. In view of these findings, and of its kinetic properties, the slow K inactivation was classified as a genuine channel gating process. The process of K activation was too fast for a dynamic analysis with the recording technique available. An estimate of its stationary voltage dependence could however be obtained in a voltage range from about -100 to about -40 mV. The experimental observations were utilized in the formulation of a mathematical model describing the kinetic behaviour of IK in the present preparation based on constant field and state transition theories.

Subthreshold and near-threshold membrane currents in lobster stretch receptor neurones.

1. The ion currents in the slowly and rapidly adapting stretch receptor neurone of lobster were investigated with respect to their nature and stationary kinetics in sub- and near-threshold voltage regions using electrophysiological and pharmacological techniques. 2. In both neurones the following currents were identified: (a) a tetrodotoxin-sensitive Na current, (b) a tetraethylammonium and 4-aminopyridine-sensitive K current, (c) a Co (or Mn)-sensitive Ca-dependent K current, (d) an ouabain-sensitive pump current and (e) a remaining leak current carried mainly by Na, K and Cl. 3. In suprathreshold voltage regions the balance between the individual membrane currents leads to the formation of a stationary negative conductance (negative slope in the voltage dependence of the ionic current) in the slowly, but not in the rapidly adapting cell. 4. These observations are compatible with the fact that during prolonged suprathreshold stimulation a stationary low frequency impulse during is possible in the slowly adapting cell, whereas in the rapidly adapting cell it is not.

Subthreshold membrane currents in slowly adapting stretch receptor neurone of lobster.

IN the slowly adapting stretch receptor neurone of lobster, application of tetrodotoxin (TTX) causes a simultaneous hyperpolarisation and decrease in input resistance1. These effects have been attributed to a possible increase in potassium conductance. They could, however, be explained by elimination of a subthreshold steady-state sodium conductance which, in this case, might underlie the cell's repetitive mode of firing2. To examine this possibility, we made a voltage clamp study of the subthreshold steady-state membrane currents of the slowly adapting stretch receptor neurone of lobster. The results indicate the existence of a subthreshold steady-state Na current which to a large extent seems to be balanced by a leak type current.

Membrane noise in slowly adapting stretch receptor neurone of lobster

ANALYSIS of membrane noise in excitable membranes may yield information about the kinetics governing the conductance control of various ionic channels. Such analysis has been applied to noise arising from conductance fluctuations in, for example, the potassium system1,2 or the system of postsynaptic ionophores3,4. This report deals with noise that seems to be generated in the sodium channel of the slowly adapting stretch receptor neurone of lobster. We recorded this noise as current noise under voltage clamp, and subsequently analysed it with respect to its covariance and spectral density function. A particularly low degree of Na inactivation at polarisations down to firing threshold was inferred from these analyses.

The Effect of Procaine on the Lowly Adapting Stretch Receptor Neurone of Lobster

The Effect of Procaine on the Lowly Adapting Stretch Receptor Neurone of Lobster

Effects of tetrodotoxin on the slowly adapting stretch receptor neurone of lobster

1. A study has been made of the effects of tetrodotoxin on the impulse activity, resting membrane potential, input resistance, and the generator potential and its after-hyperpolarization of the slowly adapting stretch receptor neurone of the lobster.2. Tetrodotoxin was able to abolish completely within about 2 min the impulse activity in most cells, when given in a dose of 2 x 10(-8) g/ml., but in all cells, when administered in a dose of 4 x 10(-8) g/ml. After blockage by the toxin in concentrations as high as 4 x 10(-6) g/ml. for periods of up to 30 min the action potential was restored by washing the preparation in physiological solution for 1 hr.3. In a concentration of 4 x 10(-8) g/ml. tetrodotoxin produced within 1-2 min an average increase of 4.8 mV of the resting membrane potential and a simultaneous 47% reduction of the resting input resistance. These effects were reversed by washing the preparation in physiological solution for 1 hr.4. Tetrodotoxin administered in doses as high as 4 x 10(-6) g/ml. for periods of up to 30 min had no effect on the amplitude of the steady phase of the generator potential.5. In a concentration of 4 x 10(-8) g/ml. tetrodotoxin produced within 5 min a 65% reduction of the amplitude of the hyperpolarization following the generator potential. This effect was reversed by washing the preparation in physiological solution for 1 hr.6. The simultaneous increase in resting membrane potential and decrease in membrane resistance is suggested to be due to an elevated potassium permeability besides a reduced sodium conductance. The constancy in height of the generator potential in the presence of a decreased membrane resistance makes necessary the assumption of an augmented generator current. The decrease in amplitude of the hyperpolarization following the generator potential may be the result of an increase in potassium conductance and/or a reduction in acceleration of an electrogenic pump in consequence of a diminished sodium influx during the generator potential.