| Popis: |
Molecule oxygen has three electronic configurations, the triplet ground state, the first and the second singlet excited states. Singlet oxygen (1O2) is highly reactive and oxidizes a wide range of biomolecules including DNA and protein. Because of the short life time in microseconds and the short working distance in nanometers, 1O2 eliminates the function of specific protein molecules at high temporal and spatial precisions and thus holds the potential as an effective photonics tool to probe protein dynamics. Recently we discovered that HCN channels are sensitive to the modification by the singlet oxygen (1O2) generated through photosensitization processes. 1O2 modification slows down the channel deactivation and enhances the expression of the voltage-insensitive instantaneous current (Iins). A histidine residue located in the pore of the HCN channel, H434, was found to be critical. The alanine replacement mutation, H434A, abolished the above effects so that 1O2 modification does not prolong the channel deactivation nor enhance the Iins. However, the reduction in Ih amplitude in H434A mutant channel becomes more significant than that in WT channel. After five short laser pulses, the amplitude of the Ih current was reduced to 76.9 ± 1.7 % of the control level in the WT channel but to 43.4 ± 2.4 % in the H434A mutant channel. This result suggests that H434 is the major target of 1O2 modification and consumes the majority of the 1O2 generated through the photosensitization process. In the H434A mutant channel, other parts of the channel reacts with the 1O2 that becomes transiently more abundant and the modification leads to the reduction in Ih amplitude. Possibly, 1O2 modification of protein molecules is not a process of random modifications but distinctly targets certain conformational and functional states. |
