Abstract
The P2X4 channel is involved in different physiological and pathological conditions and functions in the nervous system. Despite the existence of several mouse models for which the expression of the gene was manipulated, there is still little information on the expression of the protein at the cellular level. In particular, supposedly specific available antibodies have often proved to recognize unrelated proteins in P2X4-deficient mice. Here, we used an in vivo DNA vaccine approach to generate a series of monoclonal antibodies and nanobodies specific for human, mouse, and rat P2X4 channels. We further characterized these antibodies and show that they solely recognize the native form of the proteins both in biochemical and cytometric applications. Some of these antibodies prove to specifically recognize P2X4 channels by immunostaining in brain or sensory ganglia slices, as well as at the cellular and subcellular levels. Due to their clonality, these different antibodies should represent versatile tools for further characterizing the cellular functions of P2X4 in the nervous system as well as at the periphery.
Highlights
ATP is an extracellular signaling molecule that acts through two main families of plasma membrane receptors: the P2Y receptors, which are G protein-coupled receptors, and P2X receptors (P2XR), which are ATP-gated cationic channels (Burnstock, 2006)
There is a great diversity of antibody types, modes of immunization, species, and clonality, and assessing and validating their specificity is a major issue in the field (Baker, 2015; Roncador et al, 2016). This is true for polyclonal antibodies, which are produced in limited amounts and require new validation for each new batch, assuming that batches correspond to different donors. Clonal antibodies such as monoclonal antibodies or nanobodies have the advantage of being unlimited, since they are either produced by immortalized hybridoma or cloned in eukaryotic expression vectors, respectively
Immunization with synthetic peptides derived from the amino acid sequence of a transmembrane protein does not ensure proper folding corresponding to that of the native protein
Summary
ATP is an extracellular signaling molecule that acts through two main families of plasma membrane receptors: the P2Y receptors, which are G protein-coupled receptors, and P2X receptors (P2XR), which are ATP-gated cationic channels (Burnstock, 2006). In contrast to other ligand-gated channels, whose expression is mostly confined to the nervous system, P2XRs are expressed in numerous tissues and cell types (Surprenant and North, 2009). While some P2XRs are preferentially expressed in the nervous system (P2X2, P2X3), others are only found in peripheral tissue (P2X1) or are widely expressed (P2X4, P2X5, P2X7). Among all of the P2XRs, P2X4 shows the most widespread expression. It is found in diverse tissues, such as the brain, kidney, heart, lung, and salivary glands; it is expressed by many different cell types, including immune, epithelial, endothelial, and neuronal cells (Suurväli et al, 2017).
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