Zinc‐Induced Effects on Osteoclastogenesis Involves Activation of Hyperpolarization‐Activated Cyclic Nucleotide Modulated Channels via Changes in Membrane Potential

ABSTRACT Zinc is a trace element in the mammalian body, and increasing evidence shows its critical role in bone development and osteoclastogenesis. The relationships between zinc and voltage‐gated ion channels have been reported; however, the effects of zinc on membrane potential and the related ion channels remain unknown. In this study, we found that zinc‐induced hyperpolarization in RAW264.7 cells (RAW) was promoted by inhibition of hyperpolarization‐activated cyclic nucleotide modulated channels (HCNs). In electrophysiological experiments with RAW‐derived osteoclasts, HCNs were functional and generated hyperpolarization‐activated inward currents (Ih) with properties similar to the Ih recorded in excitable cells such as neurons and cardiomyocytes. Quantitative PCR of HCN subunits HCN1 and HCN4 in RAW cells showed detectable levels of HCN1 mRNA and HCN4 expression was the highest of all four subunits. HCN4 knockdown decreased osteoclastic Ih and promoted osteoclastogenesis in the presence of zinc, but not in the absence of zinc. To determine the effect of membrane hyperpolarization on osteoclastogenesis, we developed a light‐controllable membrane potential system in RAW cells by stably expressing the light‐driven outward proton pump, Archaerhodopsin3 (Arch). Arch activation by yellow‐green light hyperpolarizes the cell membrane. Light‐induced hyperpolarization accelerated osteoclast differentiation in the presence of receptor activator of nuclear factor kappa‐B ligand (RANKL). Thus, HCN activation reduced the hyperpolarization‐related promotion of osteoclast differentiation in the presence of zinc. This study revealed the novel role of HCN and membrane potential in non‐excitable osteoclasts. © 2015 American Society for Bone and Mineral Research.