Figure 4.

Deletion of αNrxns leads to reduced Ca_V2.1-mediated presynaptic Ca²⁺ influx and channel abundance. A, Isolated ω-agatoxin IVA-sensitive traces of Ca²⁺ transients through Ca_V2.1 (P/Q-type) channels derived from subtraction of transients before and after addition of the Ca_V2.1 blocker. Traces are recorded by synGCaMP6f and averaged across multiple boutons of WT, TKO, and TKO neurons transfected with Nrxn1α (TKO+Nrxn1α). B, Summary histogram of Ca_V2.1 channel contributions (in percentage of total Ca²⁺ transients) of WT boutons compared with TKO, and TKO+Nrxn1α. Data are mean ± SEM; n = ROIs/neurons (in bars), differences to WT (dotted line) are indicated above columns; ***p < 0.001; *p = 0.042, by one-way ANOVA, F_(2,1415) = 42.6. C, Representative images of immunofluorescence with antibodies against endogenous α1_A of Ca_V2.1 (magenta), colabeled against vesicular glutamate transporter (vGluT1, green) in WT and TKO neurons transfected with RFP (red) alone (WT, TKO) or in combination with Nrxn1α::GFP (cyan, TKO+Nrxn1α). Scale bar, 2.5 μm. D, Quantification of the number of α1_A-positive puncta that colocalize with presynaptic vGluT1 along RFP-filled axons of WT, TKO and TKO+Nrxn1α neurons. E, Quantification of immunofluorescence intensity of α1_A-positive puncta colocalizing with vGluT1. Data in D and E are mean ± SEM; n = number of axonal segments (shown in bars) from at least three independent experiments. **p < 0.01; ***p < 0.001, n.s. = not significant, by one-way ANOVA. F, Immunoblots of total protein lysates from two independent WT and TKO cultures, each representing ∼300,000 hippocampal cells. Blots were probed with antibodies against the α1_A pore forming and α2δ-1 auxiliary subunits of Ca_V2.1, and against all αNrxn variants that are deleted in TKO; actin = loading control.