Small conductance Ca2+-activated K+ channels (SK) channels are fundamental regulators of neuronal excitability, essential for normal neurotransmission. SK channel activity shapes interspike intervals during a burst of action potential and is thought to underlie spike-frequency adaptation. Cloned SK channels share the membrane topology of the voltage-dependent K+ channel family, but are voltage-independent, being gated solely by intracellular Ca2+ ions and thus couple intracellular Ca2+ levels and membrane potential. SK channels are heteromeric complexes, consisting of a-pore-forming subunits and calmodulin (CaM), which is constitutively bound to a 76 amino acid domain of the intracellular C-terminal region adjacent to the sixth transmembrane segment, the CaM binding domain (CaMBD). Ca2+ binding to CaM initiates structural rearrangements in the CaMBD-Ca2+ -CaM complex that are transduced into conformational alterations of the channel gate and opening of the pore. Like selective permeation, gating is a critical feature of K+ channel function. However, unlike selective permeation, a detailed structural understanding of K+ channel gating has not been obtained. Towards this goal, we have recently described the crystal structure of the CaMBD-Ca2+ CaM complex resolved to 1.60 A. Surprisingly, the structure is a dimer of two CaMBD molecules complexed to two Ca2+-CaM molecules. Based upon the crystal structure and biochemical analyses suggesting that in the absence of Ca2+ the complex exists as a monomer of one CaMBD molecule and one CaM molecule, we have presented a novel model for ion channel gating in which a Ca2+-dependent dimer-of-dimers mediates chemomechanical gating. In this application we propose to obtain a complete atomic level description of the Ca2+ dependent gating mechanism of the SK channels by solving the crystal structure of the Ca+-free form of the CaMBD-CaM complex, functionally testing the dimer-of-dimers gating model, and determining the atomic details of the interactions between the SK channel gating apparatus and a drug that modulates SK channel gating, exerting profound effects on neuronal excitability. These studies will provide a framework for understanding K+ channel gating as well as the diverse mechanisms through which CaM modulates intracellular Ca2+ levels and membrane excitability. In addition, such a detailed knowledge of the gating mechanism for SK channels will be essential for rational drug design, because modulating SK channel gating may provide a therapeutic target for pathologies of neuronal hyperexcitability such as epilepsy and schizophrenia.
| Schumacher, Maria A; Crum, Matthew; Miller, Marshall C (2004) Crystal structures of apocalmodulin and an apocalmodulin/SK potassium channel gating domain complex. Structure 12:849-60 |
