Activity-dependent endoplasmic reticulum Ca2+ uptake depends on Kv2.1-mediated endoplasmic reticulum/plasma membrane junctions to promote synaptic transmission

Abstract

The endoplasmic reticulum (ER) forms a continuous and dynamic network throughout a neuron, extending from dendrites to axon terminals, and axonal ER dysfunction is implicated in several neurological disorders. In addition, tight junctions between the ER and plasma membrane (PM) are formed by several molecules including Kv2 channels, but the cellular functions of many ER-PM junctions remain unknown. Recently, dynamic Ca²⁺ uptake into the ER during electrical activity was shown to play an essential role in synaptic transmission. Our experiments demonstrate that Kv2.1 channels are necessary for enabling ER Ca²⁺ uptake during electrical activity, as knockdown (KD) of Kv2.1 rendered both the somatic and axonal ER unable to accumulate Ca²⁺ during electrical stimulation. Moreover, our experiments demonstrate that the loss of Kv2.1 in the axon impairs synaptic vesicle fusion during stimulation via a mechanism unrelated to voltage. Thus, our data demonstrate that a nonconducting role of Kv2.1 exists through its binding to the ER protein VAMP-associated protein (VAP), which couples ER Ca²⁺ uptake with electrical activity. Our results further suggest that Kv2.1 has a critical function in neuronal cell biology for Ca²⁺ handling independent of voltage and reveals a critical pathway for maintaining ER lumen Ca²⁺ levels and efficient neurotransmitter release. Taken together, these findings reveal an essential nonclassical role for both Kv2.1 and the ER-PM junctions in synaptic transmission.

Keywords: Kv2; calcium; endoplasmic reticulum; exocytosis; synaptic transmission.

Fig. 1.

Kv2.1 has both ionotropic and nonionotropic functions in the soma. (A) Average traces of somatic QuasAr fluorescence, trial averaged from 50 AP stimulations delivered at 25 Hz. (B and C) Quantification of AP amplitude (B) and FWHM (C) (control neurons, n = 16 cells; Kv2.1 KD neurons, n = 21 cells; ***P < 0.001 for FWHM comparison, Student’s t test). (D) Example image of a cultured hippocampal neuron expressing mGreenLantern-Kv2.1. Note distinct clusters form across the membrane surface. (E) Cartoon of a neuronal soma expressing the fluorescent Ca²⁺ indicator ER-GCaMP6-150 in the ER lumen. (F) Image of the change in fluorescence of somatic ER-GCaMP6-150 in response to a train of stimulation. (G and H) Average fluorescence traces of somatic ER-GCaMP6-150 (G) and quantification of peak fluorescence (H) for both control and Kv2.1 KD neurons (control neurons, n = 12 cells; Kv2.1 KD neurons, n = 19 cells; ***P < 0.001, Student’s t test).

Fig. 2.

Endogenous Kv2.1 localizes beyond the somatodendritic compartment into axons and terminals. (A) Images of transfected then fixed DIV 16 neurons expressing GFP-Kv2.1 (cyan) and dsRedER (yellow), with immunolabeled endogenous synapsin (magenta). (B) Images of a transfected neuron expressing Ruby-Kv2.1loopBAD (yellow), 488-streptavidin (SA) (cyan), and counterstained for synapsin (magenta). The streptavidin labeling was performed on live cells before fixation and synapsin immune-detection. Yellow arrows point to colocalization of synapsin with Ruby-Kv2.1loopBAD, indicating that Kv2.1 is surface localized at presynaptic compartments. Ruby-Kv2.1loopBAD is also readily found in dendrites (blue arrow) where synapsin colocalization is absent and streptavidin labeling confirms PM insertion. A single optical section is shown. (C) Immunolabeled images of endogenous Kv2.1 (cyan) and synapsin (magenta), with merged channels (Right), in DIV 14 neurons. The center white box indicates the region enlarged as shown in the Inset. Arrows indicate Kv2.1 colocalized with synapsin-positive presynaptic terminals.

Fig. 3.

Loss of Kv2.1 impairs axonal ER Ca²⁺ influx during stimulation. (A) Image of the change in fluorescence of axonal QuasAr in response to a train of 50 stimulations delivered at 25 Hz. (B and C) Quantification of AP amplitude (B) and FWHM (C) (control neurons, n = 23 cells; Kv2.1 KD neurons n = 15 cells). (D) Cartoon of a presynaptic terminal expressing the fluorescent Ca²⁺ indicator ER-GCaMP6-150 in the ER lumen. (E) Image of the change in fluorescence of axonal ER-GCaMP6-150 in response to a train of stimulation. (F and G) Average fluorescence traces of axonal ER-GCaMP6-150 (F) and quantification of peak fluorescence (G) for control, Kv2.1 KD, and wKv2.1 rescue neurons (control neurons, n = 15 cells; Kv2.1 KD neurons, n = 24 cells; wKv2.1 rescue neurons, n = 20 cells; **P < 0.01, ***P < 0.001, Student’s t test). n.s., not significant. (H and I) Average fluorescence traces of axonal ER-GCaMP6-150 (H) and quantification of peak fluorescence (I) for both control and Kv2.1 DN neurons (control neurons, n = 5 cells; Kv2.1 DN neurons, n = 7 cells; *P < 0.05, Student’s t test).

Fig. 4.

Presynaptic Kv2.1 modulates neurotransmission independently of conduction. (A) Cartoon of a presynaptic terminal containing synaptic vesicles expressing vGlut-pHluorin, a pH-sensitive indicator of exocytosis. (B) Image of the change in fluorescence of vGlut-pHluorin in response to a train of stimulation. The arrow marks the example location of presumptive presynaptic terminals. (C and D) Average fluorescence traces of vGlut-pHluorin (C) and quantification of peak fluorescence (D) for both control and Kv2.1 KD neurons (control neurons, n = 36 cells; Kv2.1 KD neurons, n = 30 cells; ****P < 0.0001, Student’s t test). (E and F) Average fluorescence traces of vGlut-pHluorin (E) and quantification of peak fluorescence (F) for neurons ± GxTx (n = 10 cells, paired t test). n.s., not significant.

Fig. 5.

Reducing Kv2.1 expression impairs evoked presynaptic Ca²⁺ influx. (A) Cartoon of a presynaptic terminal expressing the fluorescent Ca²⁺ indicator Synaptophysin-GCaMP6f (SypGCaMP6f). (B) Image of the change in fluorescence of SypGCaMP6f in response to a train of stimulation. (C and D) Average fluorescence traces of SypGCaMP6f (C) and quantification of peak fluorescence (D) in both control and Kv2.1 KD neurons (control neurons, n = 13 cells; Kv2.1 KD neurons, n = 14 cells; *P < 0.05, Student’s t test).

Fig. 6.

The VAP-binding domain on Kv2.1 is necessary but not sufficient to rescue synaptic vesicle exocytosis. (A) Cartoon of a presynaptic terminal with endogenous Kv2.1 channels tethering the ER to the PM, positioning SERCA pumps nearby to a source of presynaptic Ca²⁺ influx. (B and C) Average fluorescence traces of vGlut-pHluorin (B) and quantification of peak fluorescence (C) in both Kv2.1 and shRNA-resistant (wobbled) Kv2.1 rescue neurons (Kv2.1 KD neurons, n = 12 cells; wKv2.1 rescue neurons, n = 14 cells; *P < 0.05, Student’s t test). (D) Cartoon of a presynaptic terminal expressing mCherry-Kv2ΔC318, which is missing the Kv2.1 VAP-binding domain and does not localize SERCA near sites of presynaptic Ca²⁺ influx. (E and F) Average fluorescence traces of vGlut-pHluorin (E) and quantification of peak fluorescence (F) in both Kv2.1 shRNA and Kv2ΔC318 rescue neurons (Kv2.1 KD neurons, n = 13 cells; Kv2ΔC318 neurons, n = 17 cells). (G) Cartoon of a presynaptic terminal expressing the CD4-Kv2.1FFAT chimera, which creates ER-PM junctions perhaps in alternate locations away from sites of presynaptic Ca²⁺ influx. (H and I) Average fluorescence traces of vGlut-pHluorin (H) and quantification of peak fluorescence (I) in both Kv2.1 shRNA and CD4-Kv2.1FFAT rescue neurons (Kv2.1 KD neurons, n = 10 cells; CD4-Kv2.1FFAT neurons, n = 11 cells).