Inactivation of the Kv2.1 channel through electromechanical coupling.

The Kv2.1 voltage-activated potassium (Kv) channel is a prominent delayed-rectifier Kv channel in the mammalian central nervous system, where its mechanisms of activation and inactivation are critical for regulating intrinsic neuronal excitability1,2. Here we present structures of the Kv2.1 channel in a lipid environment using cryo-electron microscopy to provide a framework for exploring its functional mechanisms and how mutations causing epileptic encephalopathies3-7 alter channel activity. By studying a series of disease-causing mutations, we identified one that illuminates a hydrophobic coupling nexus near the internal end of the pore that is critical for inactivation. Both functional and structural studies reveal that inactivation in Kv2.1 results from dynamic alterations in electromechanical coupling to reposition pore-lining S6 helices and close the internal pore. Consideration of these findings along with available structures for other Kv channels, as well as voltage-activated sodium and calcium channels, suggests that related mechanisms of inactivation are conserved in voltage-activated cation channels and likely to be engaged by widely used therapeutics to achieve state-dependent regulation of channel activity.

Kv2.1 expression in giant reticular neurons of the postnatal mouse brain.

Previous experiments charted the development of behavioral arousal in postnatal mice. From Postnatal Day 3 (P3) to Postnatal Day 6 (P6) mice (a) become significantly more active, "arousable"; and (b) in large reticular neurons, nucleus gigantocellularis (NGC), patch clamp recordings reveal a significantly increased ability to fire high frequency trains of action potentials as are associated with elevated cortical arousal. These action potential trains depend on delayed rectifiers such as Kv2.1. Here we report tracking the development of expression of a delayed rectifier, Kv2.1 in NGC neurons crucial for initiating CNS arousal. In tissue sections, light microscope immunohistochemistry revealed that expression of Kv2.1 in NGC neurons is greater at day P6 than at P3. Electron microscope immunohistochemistry revealed Kv2.1 labeling on the plasmalemmal surface of soma and dendrites, greater on P6 than P3. In brainstem reticular neuron cell culture, Kv2.1 immunocytochemistry increased monotonically from Days-In-Vitro 3-10, paralleling the ability of such neurons to fire action potential trains. The increase of Kv2.1 expression from P3 to P6, perhaps in conjunction with other delayed rectifier currents, could permit the ability to fire action potential trains in NGC neurons. Further work with genetically identified NGC neurons is indicated.

Single-particle cryo-EM structure of a voltage-activated potassium channel in lipid nanodiscs.

Voltage-activated potassium (Kv) channels open to conduct K+ ions in response to membrane depolarization, and subsequently enter non-conducting states through distinct mechanisms of inactivation. X-ray structures of detergent-solubilized Kv channels appear to have captured an open state even though a non-conducting C-type inactivated state would predominate in membranes in the absence of a transmembrane voltage. However, structures for a voltage-activated ion channel in a lipid bilayer environment have not yet been reported. Here we report the structure of the Kv1.2-2.1 paddle chimera channel reconstituted into lipid nanodiscs using single-particle cryo-electron microscopy. At a resolution of ~3 Å for the cytosolic domain and ~4 Å for the transmembrane domain, the structure determined in nanodiscs is similar to the previously determined X-ray structure. Our findings show that large differences in structure between detergent and lipid bilayer environments are unlikely, and enable us to propose possible structural mechanisms for C-type inactivation.