Direct current electric field induces non-linear NPFFR2 accumulation and alters its predicted conformational...

Neuropeptide FF receptor 2 (NPFFR2) has been implicated in the bioelectric response of bone marrow-derived macrophages (BMDMs). However, given the functional heterogeneity of macrophage populations, the specific response of tissue macrophages is less well characterized. In this study, we examined the regulatory effects of direct current electric field (dcEF) stimulation on NPFFR2 in primary thioglycolate-elicited peritoneal macrophages (TEPMs) by integrating cellular assays, transcriptomics, and molecular dynamics (MD) simulations. Our data indicate that dcEF intensities (25-200 mV/mm) elicited morphological polarization in TEPMs while maintaining metabolic viability. In contrast to the receptor downregulation typically observed in recruited BMDMs, dcEF exposure-particularly at 25 and 150 mV/mm-was associated with elevated NPFFR2 protein levels in wild-type TEPMs. Molecular dynamics simulations suggested that high-intensity electric fields might physically destabilize the NPFFR2 core domain, facilitating conformational unfolding. However, cellular assays concurrently revealed a net accumulation of the protein. Transcriptomic and biochemical analyses suggest that this accumulation may be attributed to a compensatory biosynthetic response, which appears to offset the heightened degradation pressure associated with structural instability. Together, these observations point to a distinct, cell-type-specific regulation of NPFFR2 by dcEF. We propose a model wherein protein accumulation in macrophages is maintained through the activation of bioenergetic and translational pathways, thereby counterbalancing field-induced physical vulnerability.