Postsynaptic densities (PSDs) were isolated from rat brain cortex and hippocampus, purified and incorporated into giant (5-80 μm in diameter) liposomes. Gigaohm seals were obtained with a patch-clamp pipette, and a giant liposome PSD-containing membrane patch, was excised and recorded. The PSD was always oriented in an inside-out configuration. This allowed receptor agonists or antagonists to be added from the interior of the recording pipette, and also the addition of different substances, such as ATP, calcium, calmodulin and others to the 'intracellular' side of the PSD, i.e. to the bath. α-Amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor agonists such as quisqualate or AMPA induced in the PSD a complex pattern of electrical activity, that was blocked by 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by 2-aminophosphonovalerate (APV). The currents generated by 0.5-1 μM quisqualate were increased by about 100% when the PSDs were phosphorylated. Similar findings were obtained when the agonist was 0.2-2 μM kainate. These currents were also blocked by a non-N-methyl-D-aspartate (NMDA) receptor antagonist but not by APV, and were increased by about 70% by phosphorylation of the PSDs. Addition of 5-10 μM NMDA plus 1 μM glycine to the 'extracellular' side of the PSD, led to a characteristic pattern of activity, with the opening of multiple receptor ion channels. This was entirely blocked by 10 μM APV. Addition of extracellular Mg2+ (1-2 mM) induced a voltage-dependent block of the currents. Phosphorylation of the PSD led to an increase of Mg2+-blocked current of about 80%. The effect of phosphorylation on ion channel activity showed a markedly different requirement for calcium and for calmodulin among the AMPA, kainate and NMDA types of glutamate receptors, thus suggesting that each receptor type is coupled at the synapse with a unique complement of protein phosphokinases.
Bibliographical noteFunding Information:
We are grateful to Drs. F. Sepúlveda and A. Stutzin for allowing us to use their patch-clamp equipments, to Dr. J.J. Marengo for making the figures, to Dr. J. Dempster, University of Strathclyde, for providing the computer software for analyzing the data, to P. Cancino for technical assistance, and to Claudia Andrade for secretarial help. Supported by Projects from Fondecyt and DTI, Universidad de Chile.
- glutamate receptors
- long-term potentiation
- patch-clamp recording
- postsynaptic densities
- protein kinase