| Popis: |
In response to both voltage and ligand, HCN channels play important physiological roles in the brain and the heart. HCN channels are activated by membrane hyperpolarization and the direct binding of intracellular cAMP. The spHCN channel was cloned from Sea Urchin and belongs to the HCN channel family. Different from the mammalian HCN1-4 channels, the spHCN channel exhibits strong inactivation in the absence of cAMP. Application to cAMP to WT spHCN channel abolishes the inactivation. Interestingly, a previously identified point mutation near the inner activation gate in S6, F459L, makes the channel behave just like the mammalian HCN channels with any inactivation. Taking advantage of the patch-clamp fluorometry technique, we set out to investigate the dynamic, activity-dependent cAMP binding during spHCN channel gating. Surprisingly, during channel activation, we observed a decrease in cAMP binding, which is directly opposite to the observation with the HCN2 channel. Conversely, in the spHCN/F459L mutant channel, we observed an increase in cAMP binding during channel activation, which is similar to that observed in HCN2 channel. These observations provide new insights into the intriguing communication between the voltage-dependent and ligand-dependent gating in HCN channels. |
