K_Ca3.1 regulates cell cycle progression by modulating Ca²⁺ signaling in murine preosteoblasts

Osteoblasts synthesize and deposit essential components of the extracellular bone matrix and collagen scaffolds, leading to mineralized bone formation. Therefore, the proliferation of preosteoblasts (precursors of mature osteoblasts) helps in regulating skeletal homeostasis. This study demonstrated that the functional expression of K_Ca3.1, an intermediate-conductance Ca²⁺-activated K⁺ channel, is markedly upregulated in murine preosteoblastic MC3T3-E1 cells in the G0/G1 phase. The enhancement of K_Ca3.1 is involved in the establishment of more negative membrane potentials in MC3T3-E1 cells. This hyperpolarization can promote intracellular Ca²⁺ signaling because store-operated Ca²⁺ channels are activated. Treatment with TRAM-34, a specific K_Ca3.1 inhibitor, attenuated the cell cycle progression from the G0/G1 phase to the S/G2/M phases. In MC3T3-E1 cells, K_Ca3.1 significantly promoted the transition from the G1 phase to the S phase. K_Ca3.1 inhibition also caused G0 phase cell accumulation. Furthermore, TRAM-34 decreased the expression of alkaline phosphatase, bone sialoprotein, and osteocalcin, osteoblast differentiation markers in MC3T3-E1 cells, and inhibited the endochondral ossification of murine metatarsals. These results reveal novel ways by which K_Ca3.1 activity can strongly modulate osteoblast maturation during bone formation.