Mapping temperature-dependent conformational change in the voltage-sensing domain of an engineered heat-activated K+ channel

Abstract

Significance Our understanding of fundamental biophysical mechanisms that underlie temperature-dependent gating remains limited despite the elucidation of structures of thermosensitive ion channels. According to current thinking, thermosensitive channels are expected to undergo a significant change in solvation upon activation by temperature, but direct evidence of such a change is lacking. Here, we utilize NMR spectroscopy and atomistic simulations to probe the solvation dynamics of an engineered temperature-sensitive variant of Shaker potassium channel at two different temperatures. Our studies reveal that this temperature-sensitive variant undergoes a dramatic shift in hydration at higher temperatures in contrast to the wild-type channels. Our findings illustrate how point mutations can cause dramatic differences in overall solvation dynamics and thereby alter temperature-dependent function. Temperature-dependent regulation of ion channel activity is critical for a variety of physiological processes ranging from immune response to perception of noxious stimuli. Our understanding of the structural mechanisms that underlie temperature sensing remains limited, in part due to the difficulty of combining high-resolution structural analysis with temperature stimulus. Here, we use NMR to compare the temperature-dependent behavior of Shaker potassium channel voltage sensor domain (WT-VSD) to its engineered temperature sensitive (TS-VSD) variant. Further insight into the molecular basis for temperature-dependent behavior is obtained by analyzing the experimental results together with molecular dynamics simulations. Our studies reveal that the overall secondary structure of the engineered TS-VSD is identical to the wild-type channels except for local changes in backbone torsion angles near the site of substitution (V369S and F370S). Remarkably however, these structural differences result in increased hydration of the voltage-sensing arginines and the S4–S5 linker helix in the TS-VSD at higher temperatures, in contrast to the WT-VSD. These findings highlight how subtle differences in the primary structure can result in large-scale changes in solvation and thereby confer increased temperature-dependent activity beyond that predicted by linear summation of solvation energies of individual substituents.