# Ion Channels and Signaling Mechanisms in T Lymphocytes

> **NIH NIH R37** · STANFORD UNIVERSITY · 2021 · $640,956

## Abstract

PROJECT SUMMARY
Signaling through store-operated Ca2+ channels (SOCs) is critical for many physiological processes including
immune cell activation and differentiation. Accordingly, the loss of SOC function leads directly to a lethal
severe combined immunodeficiency syndrome in humans. SOCs are activated by the depletion of Ca2+ from
the endoplasmic reticulum (ER), which causes the ER Ca2+sensor STIM1 to accumulate at ER-plasma
membrane (PM) junctions where it binds and activates Orai1, the pore-forming subunit of the Ca2+release-
activatedCa2+(CRAC)channel,triggeringCa2+entryintothecell.Ourlong-termgoalistounderstandin
molecular detail the underlying mechanisms that control Ca2+ influx through CRAC channels. We have
developed a number of new approaches to tackle these issues, including single-molecule tracking of STIM1
and Orai1, gene editing with CRISPR/Cas9 to label and mutagenize endogenous proteins, tandem
concatemers of Orai1 that allow subunit-selective mutagenesis of the CRAC channel, and single-molecule
FRET to probe conformational dynamics in a highly defined in vitro system. Over the next five years, we will
apply these approaches to understand CRAC channel regulation in three areas. First, we aim to understand
the mechanisms of native STIM1 and Orai1 localization and interaction at ER-PM junctions. Nearly all we
know about the SOC mechanism is based on heterologous high-level overexpression of STIM1 and Orai1,
which is likely to override many important regulatory mechanisms involving low amounts of accessory proteins.
We will exploit gene editing techniques to label and modify endogenous STIM1 and Orai1 and study the factors
that control the initial trapping of STIM1 by the PM, the residence time of Orai1 in junctions, and the
stoichiometry and interaction kinetics of STIM1 and Orai1 at native junctions. Second, we will extend
mechanistic studies of Ca2+-dependent inactivation (CDI), the predominant mechanism for feedback inhibition
of CRAC channels. By subunit-selective mutagenesis of hexameric Orai1 concatemers we will characterize
the interactions of STIM1 with the II-III intracellular loop and selected pore residues of Orai1 that drive
conformational changes underlying COl. The third and major focus will be to identify the dynamic
conformational changes that underlie activation of STIM1 and Orai1. By measuring single-molecule FRET of
labeled STIM1 and Orai1 in vitro, we will identify the structures that keep STIM1 inactive and how they
rearrange after store depletion to activate STIM1. The single-molecule approach will be widely applied to other
questions such as the stoichiometry, dynamics and conformation of STIM1 binding to Orai1 as well as the
conformational changes leading to Orai1 pore opening and the effects of purified accessory proteins thought to
modulate STIM-Orai interactions in living cells. These studies have the potential to resolve many of the most
difficult and important issues related to SOC activation, and may s...

## Key facts

- **NIH application ID:** 10217145
- **Project number:** 5R37GM045374-31
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** RICHARD S LEWIS
- **Activity code:** R37 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $640,956
- **Award type:** 5
- **Project period:** 1991-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10217145

## Citation

> US National Institutes of Health, RePORTER application 10217145, Ion Channels and Signaling Mechanisms in T Lymphocytes (5R37GM045374-31). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10217145. Licensed CC0.

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