| We have found that the conductance of GluR1i channels, as estimated from noise analysis, was agonist- and concentration-dependent. We showed that the affinity and channel open probability observed with kainate and glutamate were somewhat different, but not sufficiently so to account for the differences in observed Imax values. Spectral density analysis showed that gammanoise at saturating concentrations was 4--5 fold larger for glutamate than for kainate, which accounted for the Imax difference. However, these experiments did not explain the basis for the gammanoise differences. Single channel recordings confirmed that these channels exhibit four subconductance states that are visited in a concentration-dependent manner. Additionally, we showed, via mean low-variance and Hidden Markov analysis, that the conductance states visited are identical irrespective of agonist identity. We have also found that a point mutant, GluR1i-L497Y, formed homomeric channels that exhibited similar conductance properties as wildtype channels but were mostly resistant to glutamate-mediated desensitization. However, we found that some portion of kainate-mediated, cyclothiazide-sensitive desensitization was still intact. Additionally, we found that the channel open probability in the presence of kainate was much lower for the mutant than for the wildtype channel, and that the affinity for glutamate was 30 times smaller than the corresponding value for wildtype GluR1 channels when desensitization is intact. These data indicated that desensitization may proceed in a kinetically distinguishable manner for different agonists and that alterations in desensitization may be linked to changes in agonist binding affinity. Kinetic modeling predicted that kainate exhibits partial agonist activity because it profoundly and rapidly desensitizes the receptor, but still allows for frequent enough escapes from desensitization to appear non desensitizing. In support of this, mutant channels exhibited a channel open probability with kainate that was four-fold smaller than with glutamate. |
