C. Franke

A molecular scheme for the reaction between acetylcholine and nicotinic channels.

In outside-out patches of mouse-muscle membrane, embryonic-like channels were activated by pulses of acetylcholine (ACh). On increasing the ACh concentration, the rate of desensitization, 1/tau d, increased linearly with the peak open probability, indicating desensitization from the open state. Desensitization had only one time constant tau d at each ACh concentration. Recovery from desensitization was only approximately 10 times slower than desensitization, whereas the probability of steady-state channel opening, declined to < 0.01 with > 10(-6) M ACh. The peak probability of opening in > 10(-4) M ACh pulse was close to 1. A linear reaction scheme was not compatible with these results. The scheme had to be expanded resulting in a circular scheme with two additional ACh binding steps to desensitized channel states. The approximate rate constants of all reaction steps in the circular scheme could be determined using computer simulations. The model predicted that clusters of channel opening had the average duration tau d at the respective ACh concentration. In cell-attached patches on intact muscle fibers, similar average cluster durations were observed at the respective ACh concentration. This indicates that tau d in the intact muscle fibers has similar values as in outside-out patches.

Kinetics of homomeric GluR6 glutamate receptor channels

We studied the kinetics of the unedited version of rat GluR6 glutamate (glu) receptor channels, GluR6Q, in outside-out patches using a system for submillisecond solution exchange. Half-maximum activation of the channels was reached with approximately 0.5 microM glu. The maximum slope of the double-logarithmic plot of the peak current versus glu was approximately 1.3, indicating that at least two binding steps are necessary to open the channels. Currents in response to a pulse of 10 microM glu had a short rise time (10-90% of peak current) of approximately 220 microseconds at approximately 20 degrees C. The rise time increased with falling glu concentration, reaching approximately 6.0 ms with 10 microM glu. In the continued presence of glu, the channels desensitized, and this desensitization can be described with a single time constant of approximately 7.0 ms for a pulse of 10 microM glu. The steady-state current in response to a long pulse of 10 microM glu was below 1/280th of the peak current. The time constant of desensitization was found to be independent of concentration between 30.0 and 0.3 microM glu, but to be increased for lower concentrations. After a short pulse of 1 ms duration and 10 or 0.3 microM glu, currents decayed with a time constant of approximately 2.5 ms. Recovery from desensitization after a pulse took approximately 5 s, and the half-time of recovery was approximately 2.2 s. Continuous application of low concentrations of glutamate reduced the peak currents in response to a pulse of 10 microM glu markedly. Fifty percent response reduction was observed in the continuous presence of approximately 0.3 microM glu. Our results for homomeric GluR6 agree with a cyclical reaction scheme developed for completely desensitizing, glu-activated channels on crayfish muscles.

Open Channel Block by Physostigmine and Procaine in Embryonic‐like Nicotinic Receptors of Mouse Muscle

Embryonic‐like nicotinic channels were studied in mouse myotubes. Channel currents were measured by patch‐clamping outside‐out excised patches to which pulses of agonists and drugs could be applied by a liquid filament switch. The holding potential of the patches was generally around ‐40 mV. Pulses of 10‐4 M acetylcholine elicited average channel currents which reached a peak open probability, Po, peak, of 0.93 within 0.5 ms and decayed with a time constant of desensitization of 20‐80 ms. When physostigmine (10‐5 to 10‐3 M) or procaine (3 × 10‐5 to 10‐3 M) was added to the acetylcholine pulses, a fast decay component of the current appeared which shortened to a time constant of 0.5 ms for the maximal drug concentrations. The fast decay was followed by a slow one which declined in amplitude with increasing concentrations of the drugs. After the end of pulses of 10‐4 M acetylcholine plus 3 × 10‐4 M physostigmine the average current rose again, reaching a peak with ‐5 ms delay, and then decayed slowly. The amplitude of this recovery current was ‐0.4 Po, peak after 5 ms pulses and decreased with increasing pulse duration due to desensitization. The results can be quantitatively modelled based on a circular reaction scheme involving desensitization. Physostigmine and procaine bind to the open state to cause channel block. Also, the blocked channel was subject to desensitization. The rate constants of block were 6 × 10‐6 M‐1 s‐1 for physostigmine and 2 × 106 M‐1 s‐1 for procaine, and the rate of unblocking was 200 s‐1 for both blockers (at ‐40 mV and 20°C).

Rapid activation and desensitization by glutamate of excitatory, cation-selective channels in locust muscle

Outside-out patches of membrane were excised from extensor tibiae muscles of locusts. L-Glutamate or its agonists were applied to such patches in short pulses by means of a lipid filament switch. Cationselective, excitatory channels were activated by quisqualate, L-glutamate and aspartate (in decreasing order of effectivity), but not by ibotenate, kainate, N-methyl-D-aspartate and glycine. At high agonist concentrations, channel activation reached a peak within 1 ms. Two kinetic types of channels have been identified: L-channels with on average relatively long and S-channels with short openings. Both types of channel openings showed surprisingly high rates of desensitization, channel activity declining after the initial surge to zero with time constants of about 25 and 3 ms, respectively. The L-channels exhibit open times close to those of channels recorded in M omega-seal studies. The S-channel has not been reported previously.

Kinetic constants of the acetylcholine (ACh) receptor reaction deduced from the rise in open probability after steps in ACh concentration

Outside-out patches of enzymatically dissociated adult and denervated mouse muscle fibers were superfused repetitively by pulses of acetylcholine (ACh) containing solution. Up to 300 channels opened simultaneously 300 microseconds after the beginning of a 1,000 microM ACh pulse corresponding to a peak current i of almost -1 nA. Single responses to ACh were averaged and the concentration dependence of i and of the rise time tr from 0.1 i to 0.9 i was measured. In adult receptors, i increased proportional to the second to third power of ACh concentration, whereas in embryonic-type receptors it was proportional to the first to the second power. tr increased from approximately 0.3 ms at 1,000 microM ACh to a plateau value of approximately 5 ms for adult and of approximately 10 ms for embryoniclike receptors at concentrations less than 10 microM ACh. The concentration dependence of i and tr was simulated using the standard model of ACh binding with different combinations of rate constants and two and three binding sites for ACh. The calculated curves were compared to the measurements and a set of well fitting rate constants was determined for adult and embryoniclike receptors. Three binding sites for ACh were necessary to fit the dose response for i for adult receptors. A method for deriving rate constants in a model of ACh-receptor interaction is described that avoids analysis of open-closed kinetics of single channels, which in rapid systems, as the ones studied here, are at the limit of the frequency response of the current measurement.

Open channel and competitive block of the embryonic form of the nicotinic receptor of mouse myotubes by (+)‐tubocurarine.

1. Embryonic‐like nicotinic channels were studied in mouse myotubes. Channel currents were measured by patch clamping outside‐out excised patches to which pulses of agonists and drugs could be applied by a liquid filament switch. The holding potential of the patches was generally around‐10 to‐40 mV. 2. Pulses of 100 microM or 1 mM acetylcholine (ACh) elicited average channel currents which reached a maximum open probability of 0.93 within 0.5‐1.0 ms, decayed with a time constant of desensitization of 20‐80 ms, and fell rapidly to zero at the end of the pulse. When such pulses together with increasing concentrations of (+)‐tubocurarine (TC) were applied to outside‐out patches, the time constant of current decay, tau, decreased beginning at concentrations of TC added to the test solution of > 10 microM, and the peak amplitude of the current decreased markedly at concentrations of TC of > 30 microM due to an open channel block of nicotinic channels by TC. 3. When the outside‐out patches were pre‐incubated with TC, the peak current elicited by pulses of 100 microM ACh or 1 mM ACh + TC decreased markedly, beginning with concentrations of TC > 30 nM due to a competitive block. 4. The results could be quantitatively modelled by computer calculations based on a circular reaction scheme containing desensitization. TC blocked the open state as well as the unliganded closed state of the embryonic‐like nicotinic receptors of mouse myotubes. Also the blocked open channel was subject to desensitization. 5. The rates of block and unblock of the open channel were 3 x 10(6) M‐1 S‐1 and 0.8 S‐1, respectively, and those of the competitive block were 0.5 x 10(6) M‐1 S‐1 and 0.1 S‐1, respectively (at 20 degrees C).

Patch-clamp recordings of spiking and nonspiking interneurons from rabbit olfactory bulb slices: Membrane properties and ionic currents

SummaryPhysiological and morphological properties of rabbit, Oryctolagus cuniculus, olfactory bulb interneurons were characterized by using a thin slice preparation in combination with patch-clamp measurements and Lucifer Yellow fills. Two types of interneurons, periglomerular (PG) and juxtaglomerular (JG) cells, were unequivocally distinguished in the glomerular layer. Their properties were compared to those of mitral cells. PG cells closely resembled previously described periglomerular cells in their morphology. During current clamp recording these neurons were characterized by their lack of action potentials upon depolarization. Consistent with these results no Na+ currents could be elicited in voltage clamp experiments. Two types of outward K+ currents were distinguished: one which inactivated and one which did not. From their morphology JG cells appear to be either short axon cells or external tufted cells. JG cells always responded with a single, TTX-blockable action potential in response to maintained current injection. Two types of membrane currents were identified in JG cells during voltage clamp: a fast, inactivating Na+ current that was fully activated at — 80 mV, and a sustained outward current that shared some properties with a delayed rectifier K+ current. The particular relationship between the voltage dependence of the Na+ and K+ currents appeared to preclude repetitive spike activity.

Rapid Activation and Desensitization of Transmitter-Liganded Receptor Channels by Pulses of Agonists

The activation of transmitter-liganded receptors/channels has been studied so far mostly under steady-state conditions in the continued presence of some defined agonist concentration. Dose—response curves for the elicited current, current-noise spectra, or the open—closed kinetics of single channels have been measured in this manner, leading to elaborate schemes of the agonist—receptor reactions and the consequent opening of membrane channels (cf. Hille, 1984; Albuquerque et al., 1988; Kidekoro, 1988; Colquhoun and Sakmann, 1985; Colquhoun and Ogden, 1988).

Chloride channels gated by extrajunctional glutamate receptors (H-receptors) on locust leg muscle

Outside-out patches of extrasynaptic membrane were isolated from leg muscles of locusts. L-Glutamate and its agonists were applied to such patches either continuously or in rapidly switched pulses. When the pipette contained a high chloride concentration, 2.5 x 10(-5) M glutamate triggered single-channel currents (gated by H-receptors) with a conductance of 25 pS which were carried by chloride, in addition to cationic channels (gated by D-receptors). For the chloride channels, the distribution of channel open times had components of about 2 and 12 ms. Pulses of higher glutamate concentrations elicited many superimposed channel openings, and the approximately saturating concentration of 10(-3) M glutamate opened 100-200 channels simultaneously. When the pipette contained low chloride, channel conductance was reduced, and the current voltage relation was shifted towards the now negative chloride equilibrium potential. H-Receptor-gated chloride channels were activated by glutamate, ibotenate and aspartate, but not by GABA, quisqualate, kainate, N-methyl-D-aspartate and carbachol. The currents declined in the continued presence of agonist showing a time constant of desensitization greater than 1 s. Recovery from desensitization after removal of the agonist was tested with double pulses and was found to have a time constant of about 300 ms.

Kinetics of AMPA-type glutamate receptor channels in rat caudate-putamen neurones show a wide range of desensitization but distinct recovery characteristics

α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐type glutamate receptor channels of rat caudate‐putamen neurones were studied by ultrafast application of agonists to outside‐out vesicles taken from medium‐sized spiny neurones in thin slices. Upon application of 10 m m glutamate for 50 ms, fast rising and desensitizing currents were activated. Ten to 90% rise time values were ≈ 0.5 ms. Dose–response studies revealed an EC50 of 0.63 m m glutamate. In double logarithmic coordinates, the curve had a maximal slope between 1.33 and 1.85 at low concentrations, indicating at least two binding sites for glutamate. Rise time increased with low agonist concentrations, whereas desensitization kinetics showed only a weak dependence on concentration. The time constant of desensitization was fitted with one exponential and ranged between 2 and 11 ms, with a mean of 6.19 ± 2.31 ms (n = 239). Following brief glutamate pulses (1 ms) currents decayed with time constants of 2.7 ± 0.23 ms (n = 12). Recovery from desensitization was investigated by double‐pulse experiments. Recovery time constants fell in two subgroups with respective mean values of 110.6 ± 14.2 ms (n = 8) and 288.6 ± 33.2 ms (n = 8). By adding low glutamate concentrations to the bath solution, predesensitization of AMPA‐type receptors without channel opening could be shown. A 50% reduction in control amplitude was achieved with 5.2 ± 2.1 μm (n = 22) glutamate in the background. We hypothesize a circular reaction scheme with at least two binding sites for glutamate to describe activation, desensitization and recovery from desensitization in rat caudate‐putamen neurones.