State Dependent and Site Directed Photodynamic Transformation of HCN2 Channel by Singlet Oxygen

Abstract

Singlet oxygen (1O2) is a little known signaling factor but a useful tool for both basic research and clinical practice. 1O2 is the central player in the Chromophore Assisted Light Inactivation (CALI) for ablating the function of protein molecules or cells and the Photodynamic therapy (PDT) for treating cancer and other diseases. However, most of the studies on 1O2 have been carried out at cell and tissue levels and yielded little information about 1O2 modification at molecular level. Here we report that the hyperpolarization-activated cAMP-gated (HCN) channels are sensitive to 1O2 modification. To increase the site-specificity of 1O2 generation, fluorescein conjugated cAMP (FITC-cAMP) that specifically binds to the HCN channels or in-frame inserted singlet-oxygen-generator (SOG) protein was used as photosensitizer. Laser pulses in milliseconds transformed the channel biophysics by slowing down the channel deactivation and increasing the voltage-insensitive component in the macroscopic current. The dependence on dissolved oxygen in the solutions, the inhibitory effect by a 1O2 scavenger and the results with HCN2-SOG fusion protein supported the involvement of 1O2. Intriguingly, 1O2 modification of HCN2 channel was state-dependent and had distinct effects on the open and closed channels. Following this insight, we located a critical Histidine residue (H434) near the activation gate in the pore. Alanine replacement of H434 abolished the delay in channel deactivation and the generation of Iins by the photodynamic modification. This study provides insights into the instantaneous current conducted by HCN channel and establishes a well-defined model for studying 1O2 modification of ion channel proteins.