No abstract

Cloned NMDA receptor channels of the NRl-NR2A.NRl-NR2B and NRl-NRZC type show differences in argiotoxin,,, block. Mutations of an asparagme residue located at a homologous position in the TM2 region of all NMDA receptor subunits, which corresponds to the Q/R site of the AMPA receptors, alters the argiotoxin,,,-induced block. The results suggest that the toxin interacts at this amino acid position with the putative pore forming TM2 region of the NMDA receptor subunits. Sequence differences in the TM2 segment of NRZA and NR2C subunits are not responsible for the subtype-specific sensitivity to argiotoxin,,, as revealed by site-directed mutagenesis.

The electrophysiological analysis of Ca'+-conducting ion channels in Xenopus oocytes is difficult due to secondary intracellular effects induced by Ca 2+ . In the presence of polyvinylpyrrolidone (PVP) membrane currents can be recorded in nominally divalent cation-free solutions. The Ca2'-permeable recombinant NMDA receptors of the NRlMR2A subtype were used as assay system and the results show that PVP has no effect on NMDA receptor-induced currents. Ca" and Ba2' depress NMDA-induced currents at submillimolar concentrations probably by interfering with the Na'ZK' flux. This block is fully reversible as also observed for MC but shows in contrast no pronounced voltage dependence. PVP-containing solutions may be useful for the analysis of divalent cation-dependent ion channels.

Recombinant N-methyl-D-aspartate receptors composed of NRlNR2A subunits were expressed in Xenopus oocytes to analyse the voltage-dependent and use-dependent channel blocking activity of argiotoxin,,,. Functional assays demonstrate that the toxin competes with other open channel blockers such as MgZ+ and MK-801. Direct binding or competition assays using radiolabeled ligands and isolated rat brain membranes, in contrast, reveal no specific binding or yield binding constants which differ by orders of magnitude from the IC,, values of the functional assays. One explanation is that argiotoxin,,, does not bind with high affinity to the inhibitory site in the N-methyl-D-aspartate-receptor channel under in vitro conditions when membranes are depolarised. The structure of argiotoxin63, was investigated by NMR spectroscopy. In solution the positively charged argiotoxin,,, acquires an extended conformation and its dimensions might allow permeation deep into the channel. In the absence of direct structural information on the channel protein, the detailed analysis of blockade in conjunction with structural information, as provided here, may be of aid in the deduction of structural features of glutamate-receptor channel ion pores.