Figure 4. DAT nanodomain localization is regulated by neuronal activity in a Ca2+-dependent manner.
(A–D) Expression of the Na+ channel mNaChBac or the K+ channel Kir2.1 in DA neurons. Shown are example varicosities from DAT dSTORM of control, mNaChBac-, or Kir2.1-expressing DA neurons.
(A) DAT clusters with a diameter greater than 75 nm are shown in green and clusters smaller than 75 nm in magenta.
(B) Dense DAT clusters are shown in red (>80 localizations; radius, 50 nm).
(C) Normalized fraction of clusters greater than 75 nm (percent).
(D) Normalized fraction of DAT localizations in dense clusters (percent. Means ± S.E.; data from 3 cultures and 128 control, 290 mNaChBac, 220 Kir2.1, and 211 mNaChBac MUT varicosities; one-way ANOVA, ****p < 0.0001, **p < 0.01, *p < 0.05; n.s., not significant.
(E–J) The effect of NMDA on DAT nanodomains is blocked by the N-type VGCC blocker ω-conotoxin. Shown are example varicosities from dSTORM of DA neurons subject to 5-min treatment with vehicle, NMDA (20 μM), AP5 (100 μM) plus NMDA (20 μM), ω-conotoxin (1 μM) plus NMDA (20 μM), or ω-agatoxin (1 μM) plus NMDA (20 μM).
(E) DAT clusters with a diameter greater than 75 nm are shown in green and clusters smaller than 75 nm in magenta.
(F) Dense DAT clusters are shown in red (>80 localizations; radius, 50 nm).
(G) Localizations segmented by Voronoi tessellation.
(H and I) Normalized fraction of DAT clusters greater than 75 nm (percent) (H) and normalized fraction of DAT localizations in dense clusters (percent) (I). Means ± S.E.; one-way ANOVA, ****p< <0.0001, *p < 0.05.
(J) PDFs (curves are color coded as in the bar diagrams) for the Voronoi tessellated areas averaged by varicosity. Data are from 3 cultures and 190 control, 147 NMDA, 170 AP5, 180 ω-conotoxin, and 154 ω-agatoxin varicosities.
(K and L) Pretreatment (30 min) with BAPTA-AM inhibits the dispersing effect of NMDA on DAT nanodomains.
(K and L) Normalized fraction of DAT clusters greater than 75 nm (percent) (K) and normalized fraction of DAT localizations in dense clusters (percent) (L) for control, NMDA (20 μM) and NMDA (20 μM) plus BAPTA-AM (25 μM). Means ± S.E.; data are from 3 cultures and include analysis of 66 control, 72 NMDA, and 70 BAPTA-AM plus NMDA varicosities; one-way ANOVA, **p < 0.01, *p < 0.05.
(M) mRNA expression for P-, R-, and N-type VGCCs in DA neurons based on single-cell RNA sequencing (RNA-seq) data. Data are means ± SE of five data sets (STAR Methods).