Kamboj, Sunjeev; (1996) A patch-clamp study of native and recombinant glutamate receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The pharmacological and biophysical properties of native and recombinant non-NMDA receptors were studied using patch-clamp methods. The molecular mechanism underlying the inward rectification, which characterises Ca2+ -permeable non-NMDA receptors, was investigated. Rectification was lost during recordings from cerebellar granule cells and from isolated patches containing recombinant AMPA or kainate receptors, suggesting that it was conferred by an intracellular factor. The experiments described here suggested that intracellular spermine is the endogenous cytoplasmic factor. Thus, spermine conferred rectification in Ca2+ -permeable native AMPA receptors (from cerebellar granule cells) and recombinant AMPA receptors (composed of GluR4) and recombinant kainate receptors (formed from GluR 6(Q)/KA2). A study was made of the single channel properties of recombinant AMPA receptors containing the two alternatively spliced isoforms (flip(i) and flop(o)) of the GluR2 and GluR4 subunits, which are thought to be present in cerebellar granule cells. Several permutations were tested. The Ca2+ -permeable AMPA receptor, GluR4(i), gave conductances of ~7, 15 and 25 pS when activated by AMPA or glutamate. On the other hand, kainate gave a noise increase with no discrete resolvable events at this receptor, and an estimated conductance (from noise analysis) of ~2 pS. A similar agonist dependence of conductance has been seen in 'low-conductance' channels from cerebellar granule cells. Ca2+-impermeable (GluR2 containing) AMPA receptors had a lower conductance than GluR4(i). Receptors formed from only the GluR2 (i or o) subunits had extremely small conductances of <<1 pS. Ca2+ -impermeable heteromeric receptors formed by co-expressing GluR2 (i or o) with GluR4(i) subunits had a main conductance level of 4 pS and a sub-conductance of 8 pS when activated AMPA. These conductances resemble those activated at native low-conductance channels found in cerebellar granule cells. Some general pharmacological properties of granule cell non-NMDA receptors grown under two different culture conditions: high-K+ and low-K+, were also investigated. Their pharmacology was consistent with the expression of AMPA- rather than kainite-type receptors. The sensitivity of argiotoxin, which selectively blocks recombinant Ca2+ -permeable AMPA receptors, was correlated with Ca2+ permeability of these AMPA receptors. Ca2+ permeability also appeared to be correlated with single channel conductance of the AMPA receptors in cerebellar granule cells.

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