Structural studies of a prokaryotic cyclic nucleotide-modulated channel, MloK1, by transmission electron microscopy.

摘要

Cyclic nucleotide-modulated channel is the ion channel modulated by the binding of the cyclic nucleotides, which plays an important role in mammalian sensory transmission, heart pace regulation, and brain development. It belongs to the voltage-dependent ion channel superfamily. MloK1 is a prokaryotic cyclic nucleotide-modulated K+ channel from Mesorhizobium loti, which is a homolog of the mammalian cyclic nucleotide-modulated channel. It is found that the binding of cyclic nucleotides on the cyclic nucleotide-binding domains (CNBDs) of MloK1 promotes the ion permeation. By the determination of the MloK1 structure, we can understand how cyclic nucleotide-modulated channel activities are regulated by the cyclic nucleotide binding and how this binding associates with the channel gating. The isolation of functional MloK1 was done by protein over-expression in the Eschieria coli host, solubilization in decylmaltopyranoside (DM), and purification and concentration through gel filtration. The purified MloK1 was then two-dimensionally crystallized in the E. coli reconstituted membrane for electron crystallography.;Using transmission electron microscopy (TEM) with the techniques of image processing, such as single particle reconstruction (SPR) and Fourier filtering and unbending in electron crystallography, help us to determine the high-resolution protein structure in a "native-like" state. The full-length MloK1 three-dimensional structure was determined at 16-A resolution by negatively-stained electron microscopy and SPR, which shows a clear four-fold symmetry at an axis along the direction of pore opening when the CNBDs bind to cAMP. In electron crystallography, the two-dimensional crystal projection shows a P4212 symmetry. The homology model of MloK1 was built against the protein sequences of the transmembrane region of Kv1.2 and MloK1-CNBD and spatially constrained by the SPR model density, showing the voltage sensors are possibly in the "up"-state configuration. To understand the high-resolution details of the channel gating, cryo-electron microscopy (cryoEM) imaging of different MloK1 two-dimensional crystal forms were collected and will be analyzed in the future.