1. Departments of Physiology and Biochemistry, Howard Hughes Medical Institute, and,
2. Graduate Group in Biophysics, University of California, San Francisco, California 94143, USA
3. These authors contributed equally to this work.
4. Present addresses: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA (M.G.); Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA (H.C.L.).

Correspondence to: Lily Yeh Jan¹ Correspondence and requests for materials should be addressed to L.Y.J. (Email: lily.jan@ucsf.edu).

Abstract

Voltage-gated potassium (Kv) channels, essential for regulating potassium uptake and cell volume in plants and electrical excitability in animals, switch between conducting and non-conducting states as a result of conformational changes in the four voltage-sensing domains (VSDs) that surround the channel pore¹. This process, known as gating, is initiated by a cluster of positively charged residues on the fourth transmembrane segment (S4) of each VSD, which drives the VSD into a 'down state' at negative voltages and an 'up state' at more positive voltages². The crystal structure of Kv1.2 probably corresponds to the up state³, but the local environment of S4 in the down state and its motion in voltage gating remains unresolved^{4, 5, 6}. Here we employed several conditional lethal/second-site suppressor yeast screens to determine the transmembrane packing of the VSD in the down state. This screen relies on the ability of KAT1, a eukaryotic Kv channel, to conduct potassium when its VSDs are in the down state, thereby rescuing potassium-transport-deficient yeast⁷. Starting with KAT1 channels bearing conditional lethal mutations, we identified second-site suppressor mutations throughout the VSD that recover yeast growth. We then constructed a down state model of the channel using six pairs of interacting residues as structural constraints and verified this model by engineering suppressor mutations on the basis of spatial considerations. A comparison of this down state model with the up state Kv1.2 structure suggests that the VSDs undergo large rearrangements during gating, whereas the S4 segment remains positioned between the central pore and the remainder of the VSD in both states.

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