Abstract 1960 The N-terminal Per-Arnt-Sim (PAS) domain of hERG channels allosterically regulates channel closure and couples the voltage sensor to the pore gate

Human ether-á-go-go related gene (hERG) is a voltage-gated potassium channel that is critical for cardiac excitability. Within the KCNH channel hERG, interactions between the N-terminal Per-Arnt-Sim (PAS) of one subunit and the C-terminal Cyclic Nucleotide Binding Homology Domain (CNBHD) of a neighboring subunit has been implicated in the characteristic slow closing of the channel gate. Yet, how domains distal to the pore rearrange during gating to regulate channel the pore (and ion flux) is unclear. Here, I utilize a new and highly effective approach to photochemically crosslink hERG channels using an ultraviolet (UV) reactive ncAA benzoyl-L-phenylalanine (BZF), that can be incorporated into growing nascent peptides with TAG codon suppression technology. A pulse of UV light applied to BZF transforms it into a reversible double radical capable of forming a new carbon-carbon bond to neighboring atoms. When this is performed in tandem with electrophysiology to hold the channel in various conductive (and conformational) states, BZF can capture/isolate allosteric gating effects of domains within the channel. This approach is advantageous as BZF can i) be incorporated into proteins at locations inaccessible to metal bridging ii) provide information on interactions between regions of proteins and if they occur in a state dependent manner iii) BZF provides information on the distribution of channel conformational states under distinct voltage clamp potentials, and this is encoded within the rate and efficacy of UV crosslinking. Here, with photo crosslinking we test if the conformation of the PAS domain in hERG channels allosterically regulates the channel gate. When BZF is incorporated into the PAS domain, we observe a large change in the biophysical properties of the channel when measured with inside out patch clamp electrophysiology after photo crosslinking. When the hERG-BZF channel is irradiated, a U.V. dependent change in electromechanical coupling of the channel is observed and this shifts the voltage-dependence of conductance to more positive voltages. In addition, hERG-BZF channels closed more quickly ('Tau1' Fig4.b) compared to wild type hERG. This result indicates that during the voltage dependence of activation (when the channel transitions from closed to open) in response to membrane depolarization, the PAS domain allosterically couples the voltage sensor to the pore. In addition, while it was previously known that the PAS domain can regulate the rate to which the channel closes, here we learn that the rate of channel closure is dependent on the conformation of the PAS domain. This work is supported by NIH NIGMS MOSAIC career transition award 1K99GM144684-01A1.