Fig. 6

Transient GluA1 homotetramers form Ca²⁺ channels. a Typical time series of images of the Ca²⁺-sensitive dye Fluo-8 incorporated in the cytoplasm of HEK293 cells after l-glutamate stimulation (green images), with the images of ATTO594-labeled Halo7-GluA1 and ACP-GluA1ΔNTD expressed in the same cells (magenta images). (Top row) Two cells expressing Halo7-GluA1 at high (1.10 copies μm⁻²) and low (0.33 copies μm⁻²) number densities exhibited large or virtually no Ca²⁺ responses, respectively (typical results from 36 independent time series). (Bottom row) A cell expressing ACP-GluA1ΔNTD at a high number density (0.92 copies μm⁻²), exhibiting no changes in the cytoplasmic Ca²⁺-concentration (among 38 independent time series). Since we were unable to express ACP-GluA1 at number densities of 1.10 copies μm⁻² or higher, for these experiments, we employed Halo7-GluA1, which could be expressed up to 2.44 copies μm⁻². Dashed lines indicate the cell peripheries. b Time courses of the %increases in the Fluo-8 fluorescence intensity ([F-F₀] F₀⁻¹, where F and F₀ represent the fluorescence intensities in the cytoplasm after background subtraction, measured at times t and 0, respectively) after stimulation (time 0, vertical dotted line). The values in the graph represent the number densities of molecules expressed in the PM (copies μm⁻²). (left) The time courses for the cells on the top (orange) and bottom (cyan) shown in a. (middle) The time course for cells expressing Halo7-GluA2 at a number density of 1.18 copies μm⁻² (among 3 independent time series). (right) The time course of the cells expressing ACP-GluA1ΔNTD, shown in the bottom row in a. c [(F-F₀) F₀⁻¹]_Max (see the graph in b; mean ± SEM) plotted as a function of the number density of Halo7-GluA1 and ACP-GluA1ΔNTD expressed in the PM (graph with a bin size; 0.333 copies μm⁻²). Curves are to help the eye (fitting with quadratic functions). For the numbers of observed cells, see the circles (Supplementary Table 8). d [(F-F₀) F₀⁻¹]_Max plotted as a function of the number density of homotetramers in the PM for Halo7-GluA1 and ACP-GluA1ΔNTD. Each graph could be fitted with a linear function