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.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Channels Lacking the N-terminal Domain

“…The recently described interaction between the NR1 subunit of the NMDA receptor and EphB receptor tyrosine kinase (37), and the association of AMPA receptor subunits with Narp, a synaptic protein of the pentraxin family (18), have been reported to take place extracellularly, although the exact interaction sites have not been mapped within the iGluR ectodomains. Moreover, functional interactions of AMPA receptors with heparan sulfate containing proteoglycans present in the extracellular matrix have been suggested (38). As a large protein domain, which is likely to protrude further away from the membrane than the ligandbinding domain, the X domain would be in an ideal position to participate in the aforementioned and other protein-protein interactions.…”

Neural Cell Adhesion Molecule-associated Polysialic Acid Potentiates α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid Receptor Currents

“…For single channel analysis, data were digitally lowpass filtered at 2 kHz and compressed to final sampling rates of 20 kHz and read in pClamp software (Axon Instruments, Union City, CA). Data processing of the patch clamp recordings was essentially as described previously (41). All of the digitized traces were carefully inspected for artifacts and base-line drift before any quantitative analysis was performed.…”

“…The channel conductance was determined according to the equation g ϭ I/(V Ϫ V 0 ), where I is the current, V is the voltage, and V 0 is the reversal potential estimated from the I/V graph. To calculate the equivalent valence of the AMPA-R channel gate (z) in the presence and absence of colominic acid, we used the method described by Sinnarajah and coworkers (41) Open and closed time distributions (or the probability density functions, pdf) were assumed to be a sum of several exponential functions (i.e. pdf(t) ϭ A 1 exp(Ϫ 1 t) ϩ A 2 exp(Ϫ 2 t) ϩ A 3 exp(Ϫ 3 t), where 1 , 2 , and 3 are rate constants.…”

“…One possible explanation is that the highly polyanionic colominic acid may interact with the positively charged amino acid residues of the open channel pore of AMPA-Rs. Such a mechanism has been previously suggested for another polyanionic carbohydrate, heparin (41,50). receptor into the desensitized state can result in a decrease of interburst interval and/or increased burst duration.…”