The intracellular C-terminus confers compartment-specific targeting of voltage-gated calcium channels

Abstract

To achieve the functional polarization that underlies brain computation, neurons sort protein material into distinct compartments. Ion channel composition, for example, differs between axons and dendrites, but the molecular determinants for their polarized trafficking remain obscure. Here, we identify mechanisms that target voltage-gated Ca²⁺ channels (Ca_Vs) to distinct subcellular compartments. In hippocampal neurons, Ca_V2s trigger neurotransmitter release at the presynaptic active zone, and Ca_V1s localize somatodendritically. After knockout of all three Ca_V2s, expression of Ca_V2.1, but not Ca_V1.3, restores neurotransmitter release. We find that chimeric Ca_V1.3s with Ca_V2.1 intracellular C-termini localize to the active zone, mediate synaptic vesicle exocytosis, and render release sensitive to Ca_V1 blockers. This dominant targeting function of the Ca_V2.1 C-terminus requires the first EF hand in its proximal segment, and replacement of the Ca_V2.1 C-terminus with that of Ca_V1.3 abolishes Ca_V2.1 active zone localization and function. We conclude that Ca_V intracellular C-termini mediate compartment-specific targeting.

Keywords: CP: Cell biology; CP: Neuroscience; EF hand; active zone; axon; neuronal polarity; presynaptic; protein trafficking; synapse; synaptic vesicle; voltage-gated Ca2+ channel.

Figure 1.. Lentivirally expressed Ca_V2.1, but not Ca_V1.3, localizes to active zones and restores synaptic transmission in Ca_V2 triple knockout neurons

(A) Strategy for Ca_V2 triple knockout in cultured hippocampal neurons as described before. Transduction of neurons from triple-floxed mice with a lentivirus expressing Cre recombinase produced Ca_V2 cTKO neurons. Ca_V2 control neurons were identical except for the expression of a truncated, recombination-deficient Cre. (B) Schematic of the conditions for comparison (schematics similar to that in Held et al.); HA-tagged (HA) Ca_Vs were expressed by lentiviral transduction. (C–E) Representative images (C) and summary plots of intensity profiles (D) and peak levels (E) of Ca_V2.1 and PSD-95 at side-view synapses; levels are shown in arbitrary units (a.u.). Neurons were stained with antibodies against Ca_V2.1 (analyzed by STED microscopy), PSD-95 (STED), and synapsin (confocal). Dashed lines in (D) denote levels in Ca_V2 cTKO (black) and Ca_V2 control (gray); Ca_V2 control, 195 synapses/3 cultures; Ca_V2 cTKO, 202/3; Ca_V2 cTKO + Ca_V2.1, 205/3; Ca_V2 cTKO + Ca_V1.3, 201/3. (F–H) As in (C)–(E) but for synapses stained with antibodies against HA (to detect lentivirally expressed Ca_Vs, STED), PSD-95 (STED), and synapsin (confocal). Dashed lines in (G) denote levels in Ca_V2 cTKO (black) and Ca_V2 cTKO + Ca_V2.1 (orange); Ca_V2 control, 208/3; Ca_V2 cTKO, 222/3; Ca_V2 cTKO + Ca_V2.1, 227/3; Ca_V2 cTKO + Ca_V1.3, 214/3. (I and J) Representative traces (I) and quantification (J) of NMDAR-mediated EPSCs recorded in whole-cell configuration and evoked by focal electrical stimulation; 18 cells/3 cultures each. (K and L) As in (I) and (J) but for IPSCs; 18/3 each. Data are mean ± SEM; ***p < 0.001. Statistical significance compared to Ca_V2 cTKO was determined with Kruskal-Wallis tests followed by Dunn’s multiple comparisons post hoc tests for the proteins of interest or amplitudes in (E), (H), (J), and (L). In (H), the small decreases in HA intensity in Ca_V2 control and Ca_V2 cTKO + Ca_V1.3 compared to Ca_V2 cTKO (which does not express an HA-tagged protein) are unlikely biologically meaningful. For Ca_V C-terminal sequences and additional Ca_V expression analyses, see Figure S1.

Figure 2.. The Ca_V2.1 C-terminus suffices to target Ca_V1.3 to the presynaptic active zone

(A) Schematic of the conditions for comparison. (B–D) Representative images (B) and summary plots of intensity profiles (C) and peak levels (D) of HA and PSD-95 at side-view synapses stained for HA (STED), PSD-95 (STED), and synapsin (confocal). Dashed lines in (C) denote levels in Ca_V2 cTKO (black) and Ca_V2 cTKO + Ca_V2.1 (orange); Ca_V2 cTKO, 205 synapses/3 cultures; Ca_V2 cTKO + Ca_V2.1, 203/3; Ca_V2 cTKO + Ca_V1.3, 222/3; Ca_V2 cTKO + Ca_v1.3^2.1Ct, 218/3; Ca_V2 cTKO + Ca_V2.1^1.3Ct, 208/3. (E and F) Representative areas of confocal images (E) and quantification (F) of HA levels in synapsin regions of interest (ROIs). Identical areas (58.14 × 58.14 μm²) were imaged for confocal (E) and (F) and STED (B)–(D) analyses, and whole images were quantified in (E) and (F); 12 images/3 cultures each. Data are mean ± SEM; **p < 0.01 and ***p < 0.001. Statistical significance compared to Ca_V2 cTKO was determined with Kruskal-Wallis tests followed by Dunn’s multiple comparisons post hoc tests for the protein of interest in (D) and (F). For additional Ca_V expression analyses, see Figure S2.

Figure 3.. The first EF hand in the proximal C-terminus is essential for Ca_V2 active zone targeting

(A) Schematic of the conditions for comparison in (B)–(D). (B–D) Representative images (B) and summary plots of intensity profiles (C) and peak levels (D) of HA and PSD-95 at side-view synapses stained for HA (STED), PSD-95 (STED), and synapsin (confocal). Dashed lines in (C) denote levels in Ca_V2 cTKO (black) and Ca_V2 cTKO + Ca_V1.3^2.1Ct (purple); Ca_V2 cTKO, 207 synapses/3 cultures; Ca_V2 cTKO + Ca_V1.3^2.1Ct, 204/3; Ca_V2 cTKO + Ca_V1.3^2.1ProxCt, 209/3; Ca_V2 cTKO + Ca_V1.3^2.1DistCt, 210/3. (E) Schematic of the conditions for comparison in (F)–(H). (F–H) Representative images (F) and summary plots of intensity profiles (G) and peak levels (H) of HA and PSD-95 at side-view synapses stained for HA (STED), PSD-95 (STED), and synapsin (confocal). Dashed lines in (G) denote levels in Ca_V2 cTKO (black) and Ca_V2 cTKO + Ca_V2.1 (orange); Ca_V2 cTKO, 200/3; Ca_V2 cTKO + Ca_V2.1, 180/3; Ca_V2 cTKO + Ca_V2.1^ΔEF1, 203/3. Data are mean ± SEM; ***p < 0.001. Statistical significance compared to Ca_V2 cTKO was determined with Kruskal-Wallis tests followed by Dunn’s multiple comparisons post hoc tests for the protein of interest in (D) and (H). For additional Ca_V expression analyses, see Figure S3.

Figure 4.. Ca_V1.3^2.1Ct channels mediate neuro-transmitter release and render it L-type blocker sensitive

(A) Schematic of the conditions for comparison. (B and C) Representative traces (B) and quantification (C) of NMDAR-mediated EPSCs; 18 cells/3 cultures each. (D and E) As in (B) and (C) but for IPSCs; 18/3 each. (F) Experimental strategy to evaluate blocker sensitivity of synaptic transmission. Evoked IPSCs were recorded before blocker application (before), after wash-in of 200 nM ω-agatoxin IVA alone (+ ω-agatoxin, to block Ca_V2.1), and after wash-in of 200 nM ω-agatoxin IVA and 20 μM isradipine (+ ω-agatoxin + isradipine, to block Ca_V1s and Ca_V2.1). (G and H) Representative traces (G) and quantification (H) of IPSCs recorded as outlined in (F); 9/3 each. (I) Comparison of IPSCs normalized to “before” in each condition; 9/3 each. Data are mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001. Statistical significance compared to Ca_V2 cTKO was determined with Kruskal-Wallis tests followed by Dunn’s multiple comparisons post hoc tests in (C) and (E). Statistical significance compared to “before” was determined with Friedman tests followed by Dunn’s multiple comparisons post hoc tests in (H). Statistical significance compared to Ca_V2 control was determined with two-way, repeated-measures ANOVA followed by Dunnett’s multiple comparisons post hoc tests in (I). For additional electrophysiological data, see Figure S4. For characterization of C-terminally truncated Ca_V1.3, see Figure S5.