Heparin modulates the single channel kinetics of reconstituted AMPA receptors from rat brain

Abstract

Glutamate receptors specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have been reported to interact with the highly sulfated glycosaminoglycan, heparin, and to subsequently express lower binding affinity for [3H]AMPA. The present study examined whether heparin also modifies the kinetic properties of single channel activity expressed by isolated AMPA receptors from rat forebrain. Upon application of 280 nM AMPA, the partially purified receptors reconstituted in lipid bilayers expressed bursting channel activity that was inhibited by dinitroquinoxaline-2-3,-dione (DNQX). Treating the receptors with heparin (10 microg/ml) produced no change in conductance but the mean burst length for 280 nM AMPA was nearly doubled. Heparin also prolonged the lifetime of open states of the individual ion channels 3-5-fold, perhaps by causing a decrease in the closing rate constant for channel gating. Heparin had no effect on the lifetime of the closed state or on the amplitude of currents. The single channel open time was voltage-dependent and an increase of applied voltage caused a decrease in the heparin effect on channel open times. While the lifetime of the open channel was increased 3-4 times by heparin at 20 mV, there was no significant change induced at 43 mV. The equivalent electric charge of the channel gate was increased by 40%. The heparin effects were specific as another polysaccharide, dextran, and a monomeric constituent of heparin, glucosamine 2,3-disulfate, failed to have any effect on the receptors. These findings suggest that heparin-containing extracellular matrix components can interact with AMPA receptors and influence their functional properties.

Member of the Ampakine class of memory enhancers prolongs the single channel open time of reconstituted AMPA receptors

“…The properties of the reconstituted receptors in the present report correspond well with those described for AMPA receptors found in binding and physiological studies (Jahr and Stevens, 1987;Tang et al, 1989Tang et al, , 1991Vodyanoy et al, 1993;Wyllie and Cull-Candy, 1994;Sinnarajah et al, 1999;Smith et al, 2000). The present findings indicate that the Ampakine CX509 specifically increases the open time of AMPA receptor gated currents, and does so 25-fold more potently than aniracetam.…”

“…Panel A of Figure 1 shows trace of a recording from a patch bilayer containing AMPA receptors exposed to 150 nM AMPA with the voltage clamped at Ϫ98.7 mV. Amplitude distribution of channel currents in trace A is shown in C. Analysis of the amplitude histogram based on the longer, 20-sec trace, (data not shown) revealed that the composite current was an integral multiple of single channel currents of low and high conductance of 12.0 Ϯ 1.2 (SD) and 53 Ϯ 1.5 (SD) pS, respectively, as previously shown (Vodyanoy et al, 1993;Sinnarajah et al, 1999). Addition of CX509 resulted in a dramatic increase in the mean open time (Fig.…”

“…AMPA receptors reconstituted by the tip-dip method were activated by the addition of AMPA to the pseudoextracellular solution. The resulting current fluctuations usually had multiple conductance levels with bursts at principal conductance level of about 50 pS (Sinnarajah et al, 1999). Panel A of Figure 1 shows trace of a recording from a patch bilayer containing AMPA receptors exposed to 150 nM AMPA with the voltage clamped at Ϫ98.7 mV.…”

“…AMPA receptors from adult rat forebrain were purified with chromatographic techniques and reconstituted in small "tip-dip" bilayers as described by Bahr et al (1992). Patch-bilayers were formed in asymmetric saline conditions ("outside-out" configuration) using solution compositions reported previously (Bahr et al, 1992;Vodyanoy et al, 1993;Sinnarajah et al, 1999). After receptor incorporation, AMPA, CX509, aniracetam, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were applied at various times to the same side of the bilayer.…”

The extracellular matrix and synapses

“…Another potentially interesting link between AMPA receptors and ECM proteoglycans has been uncovered in experiments showing that heparin increases the open probability of purified AMPA receptors reconstituted in lipid bilayers (Sinnarajah et al 1999). These data suggest that members of the HSPG family directly interact with AMPA receptors to affect their activity and, possibly, stimulate their aggregation.…”