Structures and mechanisms of gating in Shaker voltage-dependent potassium channels

Ion channels, which are proteins found in the membranes of all cells, provide a mechanism for transmembrane ion flow. Shaker, a voltage-dependent potassium channel, is often used as model system for the study of ion channel structure and function.; An intracellular gate regulates the ion conduction pathway of Shaker channels in response to changes in membrane voltage. The prevailing model of gating, based on the comparison of the crystal structures of two bacterial channels, KcsA and MthK, proposes a hinge motion at a conserved glycine that splays the inner pore helices wide open. However, in contradiction to predictions made from the crystal structure of MthK, an inter-subunit metal bridge between Shaker V476C and H486 stabilizes the open state with apparently minimal distortion. Also, a metal ion can bind to multiple central facing cysteines (V474C) equally well in the open and closed states. These two types of metal bridges, which can occur simultaneously in the open state, provide tight constraints on the open Shaker channel structure. Unlike the wide-open intracellular pore entrance of MthK, these channels maintain a narrow entrance to the internal cavity even in the open state. The inner pore helices are likely to maintain the basic bundle-crossing motif of KcsA in the open state, with a bend at the conserved prolines on either side of V474C.; In a separate process, the N-terminus of Shaker causes inhibition of the current during sustained depolarization. This inactivation process is thought to act by direct blockade of the conduction pathway. We attempted to demonstrate direct interaction between cysteine mutants in the N-terminal domain and the pore domain with engineered cross-bridges. This approach did not produce any definitive evidence of interaction, perhaps due to the limited number of residues examined. However, it does not rule out the prevailing model of inactivation as direct pore blockade.; The emergence of several crystal structures of potassium channels over the last five years has provided a unique context for the study of the dynamic processes of gating. Continued dialogue between structure and function will help build our understanding of these essential proteins.