Mechanism of anion permeation through channels gated by glycine and gamma‐aminobutyric acid in mouse cultured spinal neurones.

Abstract

SUMMARY1. The ion-selective and ion transport properties of glycine receptor (GlyR) and y-aminobutyric acid receptor (GABAR) channels in the soma membrane of mouse spinal cord neurones were investigated using the whole-cell, cell-attached and outside-out patch versions of the patch-clamp technique.2. Current-voltage (I-V) relations oftransmitter-activated currents obtained from whole-cell measurements with 145 mM-Cl-intracellularly and extracellularly, showed outward rectification. In voltage-jump experiments, the instantaneous I-V relations were linear, and the steady-state I-V relations were rectifying outwardly indicating that the gating of GlyR and GABAR channels is voltage sensitive.3. The reversal potential of whole-cell currents shifted 56 mV per tenfold change in internal Cl-activity indicating activation of Cl--selective channels. The permeability ratio of K+ to Cl-(PK/PC1) was smaller than 0 05 for both channels.4. The permeability sequence for large polyatomic anions was formate > bicarbonate > acetate > phosphate > propionate for GABAR channels; phosphate and propionate were not measurably permeant in GlyR channels. This indicates that open GlyR and GABAR channels have effective pore diameters of 5-2 and 5-6 A, respectively. The sequence of relative permeabilities for small anions was SCN-> 1-> Br-> Cl-> F-for both channels.5. GlyR and GABAR channels are multi-conductance-state channels. In cellattached patches the single-channel slope conductances close to 0 mV membrane potential were 29, 18 and 10 pS for glycine, and 28, 17 and 10 pS for GABA-activated channels. The most frequently observed (main) J. BORMANN, 0. P. HAMILL AND B. SAKMANN I-> SCN-> F-for both channels. This is nearly the inverse sequence of that found for the permeability of these ions indicating the presence of binding sites for ions in the channel.8. The conductance-activity relation of GlyR and GABAR channels for Cl-was described by hyperbolic curves. The maximal conductances (Ymax) were 92 and 72 pS; the half-saturation activities (Ki) were 108 and 155 mM-Cl-for GlyR and GABAR channels, respectively. 9. The conductance-activity relation for SCN-was characterized by maximal conductances of 35 pS (GlyR) and 32 pS (GABAR). The half-saturation activities were 46 and 57 mm, respectively. In equal mixtures of SCN-and Cl-on both membrane sides the channel conductance showed anomalous mole-fraction dependence. This indicates that the channels contain at least two binding sites for anions.10. The difference between GlyR and GABAR channels in their main-state Clconductance could be caused by a small energy difference (-1 kJ mol-1) in the interaction of Cl-ions with the channel wall. The observation that both GlyR and GABAR channels display comparable conductance states, show similar permeability and conductance sequences for small anions, a similar conductance saturation curve for Cl-and SCN-and similar mole-fraction dependence of their conductance in Cl--SCN-mixtures could indicate that both receptor channels have a similar molecular st...

Agonist binding to purified glycine receptor reconstituted into giant liposomes elicits two types of chloride currents

“…It is worth noting that the probability of channel opening for the dominant and subconducting current levels in both type 'I' and type 'II' single channel responses, was increased as the holding potential was made more positive (Fig. lA), in 1(pA)15.0-, agreement with previous reports on GlyR electrical recordings in cultured cells [12].…”

“…Moreover, liposome patches responsive to the presence of agonist exhibited an intricate electrical behaviour with multiple conductance states, in agreement with the complex patterns of channel activity previously described in cultured spinal and cerebellar neurons, except that the single channel main conductance values reported in this paper are seemingly larger than most described in vivo [12,14,15].…”

“…Despite the low agonist concentration used in our experiments (considered by other authors [12,14] as non-desensitizing), the possibility of receptor desensitization (or other processes of receptor inactivation) in our system is more likely than the former explanation, as suggested by the observation of a very high activity in the initial period following seal formation and by the rapid disappearance of initial ion channel activity from several of the patches classified as 'unresponsive'.…”

A role for ion channels in glioma cell invasion

“…To the best of our knowledge, most immature cells that can migrate also accumulate intracellular Cl − and are, thus, well equipped to release KCl and to shrink, whereas there is no net force that drives Cl − out of the cell in most differentiated neurons. Because of the active accumulation of Cl − , immature neurons (Owens et al, 1996) and neuronal stem cells in the ventricular zone (LoTurco et al, 1995) depolarize in response to GABA, whereas differentiated neurons, which no longer move, either hyperpolarize or stabilize their membrane potential (Bormann et al, 1987). Although it is proposed that this depolarizing GABA response might be important physiologically in the context of synapse development (Ben Ari et al, 2004) we suggest that these cells might utilize GABA-gated Cl − channels to adjust their cell volume because they are still migrating through the brain.…”

Processing Afferent Proprioceptive Information at the Main Cuneate Nucleus of Anesthetized Cats

“…During the course of the iontophoretic experiments, mvCN neurons showed glycine sensitivity blocked by the alkaloid strychnine (see Results). The inhibitory actions of glycine receptors (GlyRs) sensitive to strychnine acting as competitive antagonist (Young and Snyder, 1973) lead to an increased chloride conductance through a ligand-gated ion channel resulting in hyperpolarization of the postsynaptic membrane (Bormann et al, 1987). The strychninebinding site of the GlyR protein complex is located in the ␣1-␣4-subunit variants, which constitute all GlyR isoforms (Becker, 1992).…”