# Molecular Dissection of Active Zone Functions in Neurotransmitter Release

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2021 · $500,407

## Abstract

Within a nerve terminal, synaptic vesicles exclusively fuse at the active zone. The active zone consists of a
protein scaffold that is anchored to the plasma membrane and forms release sites precisely opposed to
postsynaptic receptors. Interactions between active zone proteins and Ca2+ channels have long been of central
interest. Ca2+ influx through channels of the CaV2 family triggers release, and their exact positioning supports
the sub-millisecond timing of synaptic transmission and determines synaptic strength. There are two competing
models for roles and mechanisms of Ca2+ channels in synapse and active zone assembly. First, Ca2+
channels may be essential for synapse structure. Second, the active zone may recruit Ca2+ channels to release
sites, implying that synapse structure is CaV2 independent. It has been difficult to distinguish between these
models because the complexity of the Ca2+ channel gene family and their auxiliary subunits leads to extensive
redundancy. Furthermore, precisely localizing Ca2+ channels has been challenging.
We have overcome these hurdles by generating conditional triple knockout mice to remove all pore-forming a1
subunits of CaV2 channels, and by adapting superresolution microscopy to assess Ca2+ channel localization.
Our data confirm that Ca2+ flux through these channels is essential for release triggering. Based on our
preliminary data, we hypothesize that active zone assembly is independent of CaV2 channels, but instead
the active zone targets CaV2 channels with nanoscale precision to release sites. Our experimental plan
tests this hypothesis from three independent angles and dissects underlying mechanisms. In aim 1, we assess
the competing models by removing the pore forming a1 subunits, followed by assessment of synapse and active
zone structure and function. We then propose rescue experiments to assess which sequences of CaV2 channels
are required for their targeting, and we test which CaV2 sequences are sufficient to confer active zone targeting
onto non-CaV2 channels. In aim 2, we determine the precise presynaptic localization of auxiliary subunits and
assess whether their presynaptic targeting depends on a1. We then test whether functional roles of these
auxiliary subunits require the presence of a1. In aim 3, we address molecular mechanisms for CaV2 targeting
from the perspective of active zone scaffolds. We first determine the order of arrival of active zone and CaV2
proteins during active zone assembly, and we then determine localization and function of CaV2s and their
subunits in mutants that lack specific active zone proteins.
This grant will test two fundamentally different models of the relationship between Ca2+ channels and the active
zone, and dissects the mechanisms that underlie Ca2+ channel anchoring at the target membrane. Precise
understanding of these mechanisms is important for understanding synapses in health and disease.

## Key facts

- **NIH application ID:** 10145802
- **Project number:** 5R01NS083898-08
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Pascal Simon Kaeser
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $500,407
- **Award type:** 5
- **Project period:** 2014-07-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10145802

## Citation

> US National Institutes of Health, RePORTER application 10145802, Molecular Dissection of Active Zone Functions in Neurotransmitter Release (5R01NS083898-08). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10145802. Licensed CC0.

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