Modulating ion channels with nanobodies

Abstract

Ion channels play instrumental roles in regulating membrane potential and cross-membrane signal transduction, thus making them attractive targets for understanding various physiological processes and associated diseases. Gaining a deeper understanding of their structural and functional properties has significant implications for developing therapeutic interventions. In recent years, nanobodies, single-domain antibody fragments derived from camelids, have emerged as powerful tools in ion channel and synthetic biology research. Their small size, high specificity, and ability to recognize difficult-to-reach epitopes offer advantages over conventional antibodies and biologics. Furthermore, their resemblance to the variable region of human IgG family III reduces immunogenicity concerns. Nanobodies have introduced new opportunities for exploring ion channel structure-function relationships and offer a promising alternative to conventional drugs, which often face challenges such as off-target effects and toxicity. This review highlights recent progress in applying nanobodies to interrogate and modulate ion channel activity, with an emphasis on their potential to overcome current technical and therapeutic limitations.

Keywords: Antibody engineering; Immunotherapy; Ion channels; Nanobody; Synthetic biology; Therapeutics.

Graphical abstract

Fig. 1

Schematic illustration of human and camelid antibodies and their fragments. In a typical mammalian antibody, the light chain consists of one variable (VL, light grey) and one constant (CL, dark grey) domain, while the heavy chain contains one variable (VH, purple) and three constant (CH1 to CH3, dark grey) domains. The antigen binding fragment (F_ab) is formed by the paring of VL and VH domains. In contrast, the camelid antibody exists as a homodimer with heavy chains only. The isolated variable region of the camelid antibody provides a functional single-domain antibody (VHH), commonly known as a nanobody.

Fig. 2

Schematic diagram showing the generation of P2X7-targeting nanobodies. A. Llama was immunized with human and mouse P2X7-transfected HEK293 cells to generate anti-P2X7 nanobodies. B. Nanobodies were isolated as monomers and re-engineered into dimer along with Alb8. C. Nbs inhibit ATP-activated P2X7 channels, which are involved in inflammatory response by inducing Ca²⁺ influx and ultimately causing the production of pro-inflammatory cytokines, such as IL-1β.

Fig. 3

Nanobody-mediated degradation of Ca_V channels. A. The anti-Ca_Vβ nanobody nb.F3 is fused with the catalytic HECT domain of the Nedd4L E3 ubiquitin ligase to generate Ca_V-aβlator. Ca_V-aβlator is capable of catalyzing the ubiquitination of the Ca_V1.2/Ca_Vβ Ca²⁺ channel, ultimately leading to their functional inhibition. B. The anti-Ca_Vβ1 nanobody nb.E8 is fused with the catalytic HECT domain of Nedd4L to yield Chisel-1. Chisel-1 catalyzes the ubiquitination of the Ca_V1.2/Ca_Vβ1 Ca²⁺ channel to suppress the channel activity via proteasomal degradation.

Fig. 4

Nanobody-mediated direct modulation of ASICs or Na_V channels. A. Cartoon illustrating how Nb.C1 interferes with the binding of a venom toxin from the Texas coral snake (MitTx), thereby inhibiting MitTx -activated hASIC1a channels. B. A Nb.C1-PcTx1 fusion protein is engineered to provide more precise hASIC1a targeting with an enhanced analgesic effect. C. Nb17 and Nb82 specifically target the C-terminal regions of the 1.4 and 1.5 isoforms of Na_V channels for functional tuning.