Single Particle Image Reconstruction of the Human, Recombinant Kv2.1 Channel

¹ The Scripps Research Institute
² Glaxo Wellcome Research Institute
³ University of Chicago

^* To whom correspondence should be addressed. E-mail: yeager{at}scripps.edu.

Submitted on August 7, 2007
Revised on September 13, 2007
Accepted on 11 December 2007

	Abstract

Kv2.1 channels are widely expressed in neuronal and endocrine cells and generate slowly activating K⁺ currents, which contribute to repolarization in these cells. Kv2.1 is expressed at high levels in the mammalian brain and is a major component of the delayed rectifier current in the hippocampus. In addition, Kv2.1 channels have been implicated in the regulation of membrane repolarization, cytoplasmic calcium levels, and insulin secretion in pancreatic cells. They are therefore an important drug target for the treatment of Type II diabetes mellitus. We used electron microscopy and single particle image analysis to derive a three-dimensional density map of recombinant human Kv2.1. The tetrameric channel is egg-shaped with a diameter of ~80 Å and a long axis of ~120 Å. Comparison to known crystal structures of homologous domains allowed us to infer the location of the cytoplasmic and transmembrane assemblies. There is a very good fit of the Kv1.2 crystal structure to the assigned transmembrane assembly of Kv2.1. In other low-resolution maps of K⁺ channels, the cytoplasmic N-terminal and transmembrane domains form separate rings of density. In contrast, Kv2.1 displays contiguous density that connects the rings, such that there are no large windows that connect the channel interior to the cytoplasmic space. The crystal structure of KcsA is thought to be in a closed conformation, and the good fit of the KcsA crystal structure to the Kv2.1 map suggests that our preparations of Kv2.1 may also represent a closed conformation. Substantial cytoplasmic density is closely associated with T1 and is ascribed to the ~184kDa C-terminal regulatory domains within each tetramer.

Key Words: Potassium channels, electron microscopy, image analysis, voltage-gated channels

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