Mechanisms of regulation of hERG ion channels by cytoplasmic factors

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Ion channels control membrane excitability and are critical determinants of cellular function. Voltage-gated K (Kv) channels, the largest sub-family, are responsive to the transmembrane potential and regulate excitability in cardiac, nerve, and endocrine tissues. In cardiac tissues, opening of Kv channels dampens excitability and terminates the action potential. The human ether-a-go-go related gene (hERG) Kv channel is a critical player mediating cardiac repolarization and this is highlighted by the association of hERG dysfunction with cardiac arrhythmia. hERG channel structure is similar to that of other Kv channels: S1-S6 transmembrane domains with S1-S4 forming the voltage sensor and S5-S6 the pore. S4 contains basic amino acids that sense transmembrane voltage and trigger movement of S4, which opens the pore. Coupling of S4 movement to the pore involves a short, but important, cytoplasmic S4-S5 linker. hERG channels also possess an N-terminal Per-Arnt-Sim (PAS) domain and C-terminal cyclic nucleotide binding domain (cNBD). In contrast to other Kv channels, the gating properties of hERG channels are unique and poorly understood. This affords them their critical role in termination of the action potential. hERG channels activate and deactivate slowly, yet inactivate and recover from inactivation rapidly. Because inactivation is faster than activation, hERG currents during depolarization (early in the action potential) are small, and robust current is only observed upon repolarization (action potential termination) when channels rapidly recover from inactivation, but have yet to close. The slow closing is thus a crucial feature that allows current to flow during repolarization and this is highlighted by the association of mutations that accelerate deactivation gating with cardiac arrhythmia. Despite this, the mechanistic basis of these unusual gating events is poorly understood. Perturbations in hERG gating occur in response to mutations in different regions of the channel: N- and C-terminus, voltage sensing unit and S4-S5 linker. However, the mechanism by which these signals from diverse regions are integrated is unclear. We propose that the cytoplasmic S4-S5 linker plays a key role as an integrator of signals from cytoplasmic channel domains, i.e. N- and C-terminal domains would modify gating by direct interaction with the S4-S5 linker that modulates S4 movement or its coupling to the pore gate. We propose that the S4-S5 linker may integrate numerous cytoplasmic signals in this way. We propose a combined approach of electrophysiology, hypothesis-driven mutagenesis and biochemistry to investigate how hERG function is controlled by cytoplasmic domains of the channel. In Objective 1, we propose to define interactions between the S4-S5 linker and N- and/or C- terminus that regulate hERG channel function. We hypothesize that the S4-S5 linker makes specific key contacts that mediate interaction with one/both of these domains and we will use site-directed mutagenesis and two-electrode voltage clamp to test this. In Objective 2, we propose to measure these interactions directly using isothermal titration calorimetry to detect bi-molecular protein-protein interactions. This approach allows for biophysical measurements of the reaction to inform on the nature of the interactions. Lastly, in Objective 3, we propose to determine the physiological role of the N-terminal PAS domain in hERG channels. Its physiological role in hERG channels is not known, and we intend to investigate a putative role in oxygen and/or redox potential sensing and its transduction to hERG channel functional changes. Thus, our combined technical approach aims to describe the molecular mechanisms by which hERG channels are gated by cytoplasmic signals.