# Development of a nanobody tool kit to study connexin channels

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2024 · $201,250

## Abstract

There are 21 different connexin isoforms in human that play crucial roles in intercellular
communication during development and physiology. At molecular level, six connexin proteins
assemble as hexamers to form an hemichannel that is inserted in the plasma membrane. The
docking of two hemichannels from adjacent cells form a gap junction channel (GJC); both
hemichannels and GJCs are permeable to ions and small metabolites (<1 kDa). Several human
connexin mutations and multiple pathological conditions exacerbate the activity of undocked
hemichannel causing cellular dysfunction. A major limitation in the field is the lack of adequate
pharmacological and genetic tools to dissect out the role of undocked connexin hemichannels
versus their functions as GJCs. The objective of this proposal is to create recombinant
nanobodies targeting Cx43 protein that differentially recognize hemichannels from GJCs to
overcome this technical barrier. These nanobodies have reduced size (15 kDa) allowing higher
tissue penetration, reduced immunogenicity and easy genetic manipulation and engineering. To
achieve this goal, we have selected peptides corresponding to extracellular regions, N-terminal
helix and intracellular loop of Cx43 protomer for llama immunization. To identify and generate
recombinant Cx43 nanobodies present in this serum, we will use the immunized llama’s B-cells
as source of nanobodies-coding RNA to produce a nanobodies-phage display library. The
selected nanobodies will be screening to recognize Cx43 via ELISA, Western blot and
immunofluorescence. In addition, we will assess the nanobodies capabilities to block Cx43
hemichannel activity in vitro and their wide applicability using heterologous expression system
and disease models. Considering the compelling evidence for a pathological role of Cx43
hemichannels in various diseases and our willingness to share the resulting antibodies with the
whole scientific community, the present project is highly innovative and significant.

## Key facts

- **NIH application ID:** 10952384
- **Project number:** 1R21GM155757-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** Jorge Enrique Contreras
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $201,250
- **Award type:** 1
- **Project period:** 2024-09-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10952384

## Citation

> US National Institutes of Health, RePORTER application 10952384, Development of a nanobody tool kit to study connexin channels (1R21GM155757-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10952384. Licensed CC0.

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