Structural organization of gap junction channels

AbstractGap junctions were initially described morphologically, and identified as semi-crystalline arrays of channels linking two cells. This suggested that they may represent an amenable target for electron and X-ray crystallographic studies in much the same way that bacteriorhodopsin has. Over 30 years later, however, an atomic resolution structural solution of these unique intercellular pores is still lacking due to many challenges faced in obtaining high expression levels and purification of these structures. A variety of microscopic techniques, as well as NMR structure determination of fragments of the protein, have now provided clearer and correlated views of how these structures are assembled and function as intercellular conduits. As a complement to these structural approaches, a variety of mutagenic studies linking structure and function have now allowed molecular details to be superimposed on these lower resolution structures, so that a clearer image of pore architecture and its modes of regulation are beginning to emerge

Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments1 or require light and are difficult to use2. Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed3,4. MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix

Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments[superscript 1] or require light and can be difficult to use[superscript 2]. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed[superscript 3, 4]. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen.National Institutes of Health (U.S.) (Grant DP1 OD003961)National Science Foundation (U.S.). Graduate Research Fellowship ProgramUnited States. Dept. of Defense (National Defense Science and Engineering Graduate (NDSEG) Fellowships

Three-Dimensional Structure of the Gap Junction Connexon

The gap junction membrane channel is composed of macular aggregations of intercellular channels permitting the direct intercellular transfer of ions and small molecules. Each intercellular channel is formed by the apposition of two hexameric transmembrane channels (connexons), one from each cell. The interlocking of the two channels occurs extracellularly in a narrow 3.5-nm “gap” separating the junctional membranes. The channel-channel interaction is known to be selective between members of the family of proteins, called connexins, which oligomerize into the connexons. In addition to selectivity, the molecular interfaces involved in the extracellular interactions between connexons must be very congruent, since the intercellular channel must provide high resistances to the leakage of small ions between the channel lumen and the extracellular space. By using a recently developed biochemical procedure for obtaining ordered arrays of connexons from gap junctions split in the extracellular gap, (Ghoshroy, S., D. A. Goodenough, and G. E. Sosinsky. 1995. Preparation, characterization, and structure of half gap junctional layers split with urea and EGTA. J. Membr. Biol. 146:15-28) a three-dimensional reconstruction of a connexon has been obtained by electron crystallographic methods. This reconstruction emphasizes the structural asymmetry between the extracellular and cytoplasmic domains and assigns lobed structural features to the extracellular domains of the connexon. The implication of our hemichannel structure is discussed in relation to the in vivo state of unpaired connexons, which have been shown to exist in the plasma membrane