(P30-028-25) Vitamin K2 Attenuates Calcium Influx via Cav1.2 Channels to Modulate Muscle Contraction

Objectives: To investigate the mechanism by which Vitamin K2 exerts its antispasmodic effect on muscle cells.

Methods: 1. Construction of tool cells expressing Cav1.2, Kir2.1, and KCa1.1.

Genes encoding Cav1.2, Kir2.1, and KCa1.1 were cloned from the human genome. Expression vectors were transfected into CHO cells (targeting Cav1.2 and KCa1.1) and HEK293 cells (targeting Kir2.1) via electroporation.

2. Effects of Vitamin K on ion channels in tool cells

CHO cells expressing Cav1.2 or KCa1.1 and HEK293 cells expressing Kir2.1 were treated with Vitamin K1, Vitamin K2, and Vitamin K3. Channel inhibitors included Nifedipine for Cav1.2, BaCl2 for Kir2.1, and TEA-Cl for KCa1.1 were used as positive control. Changes in current after following treatment with Vitamin K or channel inhibitors were recorded using patch-clamp techniques.

3. Effects of Vitamin K2 on ion channels in smooth muscle cells:

Rat aortic smooth muscle cells (A7r5) were treated with different conditions: Cav1.2 channel agonist (S)-(-)-Bay-K-8644, (S)-(-)-Bay-K-8644 + Vitamin K2, and (S)-(-)-Bay-K-8644+ Vitamin K2+ Warfarin. Changes in calcium ion currents were recorded using patch-clamp techniques.

Results: 1. qPCR analysis and patch-clamp recordings validated the successful expression and functional activity of Cav1.2 and KCa1.1 proteins in CHO cells, as well as the Kir2.1 protein in HEK293 cells.

2. Vitamin K3 showed no significant effect on Cav1.2, Kir2.1, or KCa1.1 channels. Vitamin K1 displayed mild inhibitory activity on Cav1.2, reducing its activity by 15.74% ± 5.92%. Meanwhile, Vitamin K2 showed a time-dependent and substantial inhibitory effect on Cav1.2, achieving a maximum inhibition of 82.32% ± 2.91% at 600 seconds.

3. Vitamin K2 did not inhibit currents in resting A7r5 cells but effectively suppressed calcium influx upon activation by the Cav1.2 channel agonist (S)-(-)-Bay-K-8644. Notably, this inhibitory effect remained unaffected by Warfarin.

Conclusions: Vitamin K2 directly inhibits calcium influx through Cav1.2 channels. This inhibitory effect is observed following activation of Cav1.2 channels by agonists and occurs independently of carboxylation of Vitamin K-dependent protein.

Funding Sources: This work was supported by Sichuan Province Science and Technology Plan Key Research and Development Project (24DYF1223)，