Photodynamic Modification of Native HCN Channels in Thalamocortical Neurons

Abstract

Previously, we reported photodynamic modification (PDM) of heterologous expressed HCN channels on membrane patches. In the presence of specific (FITC-cAMP) and non-specific (Rose Bengal) photosensitizers, light excitation transforms the function of HCN channels in an oxygen dependent manner. Here we extended the study to native HCN channels expressed in thalamocortical (TC) neurons in the Ventrobasal (VB) complex in the thalamus. We first discovered that blue light excitation significant increases the current amplitude and the rate of activation of the hyperpolarization-activated Ih current, which was a reversible process and did not require exogenous photosensitizers. However, when FITC-cAMP, but not FITC alone or FITC and cAMP, was loaded into the cell through whole-cell recording pipette, light excitation resulted in long-lasting increases in the voltage-insensitive, instantaneous Iinst component and correspondingly decreases in the Ih component. The Ih and Iinst after PDM can be blocked by Cs+ and ZD7288, confirming the specific involvement of HCN channels. Next, we investigated the impacts of PDM of native HCN channels on the resting membrane potential (RMP) and input resistance (Rin) of VB neurons. As expected, after PDM, there was a significant positive shift in RMP. Importantly, both the long-lasting increase in Iinst and the positive shift in RMP after light excitation, but not the short-term and reversible increase in Ih during light excitation, could be blocked by Trolox-C, a widely used quencher for singlet oxygen. In summary, we report the PDM of native HCN channels under more physiological condition which should carry meanings for both basic researches and clinical treatments of diseases due to HCN channelopathy.