Electron microscopic image analysis of cardiac gap junction membrane crystals

Abstract

Cardiac gap junctions play an important functional role in the myocardium by electrically coupling adjacent cells, thereby providing a low resistance pathway for cell-to-cell propagation of the action potential. Two-dimensional crystallization of biochemically isolated rat ventricular gap junctions has been accomplished by an in situ method in which membrane suspensions are sequentially dialyzed against low concentrations of deoxycholate and dodecyl-beta-D-maltoside. Lipids are partially extracted without solubilizing the protein, and the increased protein concentration facilitates two-dimensional crystallization in the native membrane environment. The two-dimensional crystals have a nominal resolution of 16 A and display plane group symmetry p6 with a = b = 85 A and gamma = 120 degrees. Projection density maps show that the connexons in cardiac gap junctions are formed by a hexameric cluster of alpha 1 connexin subunits. Protease cleavage of alpha 1 connexin from 43 to 30 kDa releases approximately 13kDa from the carboxy-tail, and the projection density maps are not significantly altered. Uranyl acetate stain penetrates the ion channel, whereas phosphotungstic acid is preferentially deposited over the lipid regions. This differential staining can be used to selectively probe the central channel of the connexon and the interface between the connexon and the lipid. The hexameric design of alpha 1 connexons appears to be a recurring quaternary motif for the multigene family of gap junction proteins.

Cell-free synthesis and assembly of connexins into functional gap junction membrane channels

“…Electron diffraction analysis of isolated GJ plaques (Unwin and Zampighi, 1980; Yeager, 1995; Unger et al ., 1997) and crosslinking of isolated GJ connexons (Cascio et al ., 1995) have indicated that a GJ connexon is composed of six subunits. To determine if the GJ channel subunit proteins that were synthesized in the cell‐free system assembled into oligomers, microsomes derived from an α 1 fl and a β 1 fl translation reaction were solubilized in 1% Triton X‐100 (TX‐100), and the Cx translation products were separated by sucrose gradient sedimentation analysis as described in Materials and methods.…”

“…generated that no longer contained the specific epitopes recognized by the α 1 S-and β 1 S-specific monoclonal Assembly of cell-free synthesized connexins into GJ connexons antibodies (α 1 ΔC and β 1 ΔC, Figure 2D). Co-translation of the full-length connexins (α 1 fl or β 1 fl), together with Electron diffraction analysis of isolated GJ plaques (Unwin and Zampighi, 1980;Yeager, 1995;Unger et al, 1997) their corresponding truncated connexins (α 1 ΔC or β 1 ΔC) in the presence of microsomes, resulted in the and crosslinking of isolated GJ connexons (Cascio et al, 1995) have indicated that a GJ connexon is composed of immunoprecipitation of the full-length connexins and also in the co-precipitation of the truncated Cx polypeptides. six subunits.…”

High-Throughput, Algorithmic Determination of Nanoparticle Structure from Electron Microscopy Images

“…Recent computational methods have begun to address these limitations through the automated identification of classes of nanoparticle shapes or the determination of a single structural parameter ( e.g. , size) for limited cases of homogeneous nanoparticle populations. ,− However, these methods lack the structural detail and shape specificity required to characterize a nanoparticle population in a way that correlates with ensemble measurements. Herein, we describe a high-throughput, quantitative, structurally specific computational analysis of EM images that allows us to relate a small sample (relative to the total number of particles) to the global properties of heterogeneous populations.…”