I_k Potassium Current And Low Potassium Induced Apoptosis In Rat Cerebellar Granule Cells Of The Relationship

Apoptosis, the programmed cell death, plays a critical role in development and physiological function of multi-cellular organisms. The abnormal regulation of cell death can cause serious diseases such as cancer, hypertension, cardiopathy, virus infection, immunological and nerve degeneration related diseases. Intracellular K concentration has a great effect on the generation and development process of apoptosis. Extensive researches have shown that in Caspases-dependent apoptosis with DNA fragment production, the potassium outward influx increases and intracellular potassium concentration decreases in the early process of apoptosis. Among so many different kinds of potassium channels, the delayed rectifier potassium channel (I_K) has been proposed to play the most important role in enhancement of potassium outward current during the early part of apoptosis due to its high conductance for potassium ions and none or slow inactivation. However, the mechanism of the action and modulation of the delayed rectifier potassium channel is still unknown. Therefore, we used electrophysiological and molecular biochemical techniques to study the mechanism of interaction between I_K type potassium channel and apoptosis and the possible channel subunit related to apoptosis in mouse cerebellar granule cells which are treated with low potassium.First, with whole cell patch clamp configuration, we found that the magnitude of I_K increased greatly and the kinetics of the channel changed after incubation of granule cells with 5mM potassium culture solution for 6 hours while the potassium concentration in normal physiological culture solution is 25mM. With RT-PCR technique, we found that the intracellular level of mRNA of Kv2.1 subunit, the major component of I_K channel, increased significantly after apoptosis. And the expression of Kv2.1 protein in the membrane increased with the time too. After that, the low livability and DNA fragmentation happened, which indicated apoptosis of the cell. TEA, the I_K channel blocker, could inhibit the amplitude of I_K current and protect cell from apoptosis induced by low potassium. Decrease of Kv2.1 mRNA intracellular level with RNA interference also inhibited the amplitude of I_K current and increasedcell livability. Overexpression of Kv2.1 protein enhanced the generation of apoptosis in the granule cell. Our data indicated that the outward I_K current due to activation of I_K channel was an important factor for the apoptosis induced by low potassium.The previous data in our lab showed that melatonin could regulate I_K channel bilaterally through different types of receptors in rat granule cells. Then we examined the effects of 2-iodomelatonin, an activator of melatonin receptor, on the apoptosis. Our results showed that 2-iodomelatonin inhibited I_K currents amplitude and changed its kinetics. 2-iodomelatonin increased the cell livability after low potassium induced apoptosis and inhibited the activity of Caspase-3 which is a key element for apoptosis. Unlike melatonin, 2-iodomelatonin is not an antioxidant. The protective effect for the cell and inhibition of the activity of Caspase-3 by 2-iodomelatonin was blocked by 4P-PDOT, an antagonist of melatonin receptor. This indicated that 2-iodomelatonin could inhibit apoptosis by activation of melatonin receptor and inhibition of I_K channel subsequently but not mediated through anti-oxidization effect.Signal transduction pathways, such as PKA and PKC pathway, also play a critical role in apoptosis. However, the interaction between ion channel and signal transduction is unclear. Our results showed that forskolin, an adenylyl cyclase activator, decreased the amplitude of I_K current in granule cell instantly and change the kinetics of the channel, which was blocked by an inhibitor of PKA, H-89. Moreover, incubation with forskolin decreased the intracellular level of Kv2.1 mRNA and changed the kinetics of the channel. Further experiments showed that forskolin increased the livability of granule cell after apoptosis and inhibit DNA fragmentation, which was blocked by H-89. Decreased intracellular level of Kv2.1 mRNA with RNA interference inhibited the decrease of I_K current caused by forskolin and its protective effect for granule cell from apoptosis. These results indicated that I_K channel, especially Kv2.1 subunit, is an important downstream factor for the protection of cells from apoptosis by PKA pathway.Recent studies showed that the alteration of lipid components in the membrane could influence the distribution and function of ion channels. Thus, we examined the regulation of I_K channel and apoptosis by cholesterol. The concentration change ofcholesterol in the membrane affected the amplitude and kinetics of I_K, changed the distribution of channel proteins and decreased the sensitivity of induction of apoptosis in granule cell. These primary results indicated that the effect of cholesterol on apoptosis could involve in regulation the I_K current. The mechanism of the interaction and regulation will be explored in the future experiment.