| Summary: | 碩士 === 國立陽明大學 === 生理學研究所 === 89 === Overexpression of an aberrant gene of voltage-gated K channel (Kv3.4) mRNA and protein has been reported in human oral squamous cell carcinoma (OSCC). In additions, A-type K channel blocker, 4-AP, and antisense oligonucleotids of Kv3.4 both caused a significant reduction of OSCC cell proliferation apparently due to the induction of apoptosis. Electrophysiological study on programmed cell death has revealed that apoptotic proteins (such as reaper, grim, or hid) may participate in initiating apoptosis by irreversible blocking K channels via "ball and chain" inactivation fashion. Moreover, the results of the study structure-function basis of phospho-regulation of inactivation gate by using synthetic inactivation domain have shown that the N-type inactivation of Kv3.4 is dynamically regulated either by protein phosphorylation or by oxidizing reagents. We, therefor, have hypothesized that the removal of inactivation of Kv3.4 is required in the process of proliferation in oral cancer cell lines (OEC-M1) under reactive oxygen species (ROS) stress. The gating kinetics of OEC-M1, with or without extra Kv3.4 gene, and normal human oral keratinocyte (NHOK) were studied by using patch clamp whole cell recording techniques. Although we are unable to dissect A-type current from capacitive current in some NHOK and OEC-M1 patches due to the fast activating kinetics of A-type K current. Much to our surprise is that the chance of observing A-type K channel in OEC-M1 is lower than that of NHOK. NHOK has a linear I-V relationship, whereas that OEC-M1 transected with extra Kv3.4 gene has an outward rectifying I-V relationship. The inactivation kinetics in NHOK is fast (t = 2 ~ 3 msec) compare to those of OEC-M1 transected with extra Kv3.4 gene (t = 10 ~ 40 msec), however, two types of inactivation kinetics: fast (t = 1 ~ 3 msec) and slow (t = 20 ~ 40 msec), were seen in OEC-M1. Furthermore, the reducing agent, b-mercaptoethanol, does slow the inactivation kinetics in a voltage-dependent fashion. These results are consistent with our hypothesis yet no proof at this moment. The preliminary results of our patch-clamping study have suggested that the gating kinetics of Kv3.4 channel may play an important role in cell proliferation and programmed cell death. The goal of that whether the oncogenic potentials of the Kv3.4 channels may be mediated by immobilization of the inactivation ball domain still need to be proceeded.
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