Figure 4.2. Ultrastructure of natural and purified gap junctions.
The figure below (Figure 4.3) diagrams the components of the gap junction and how they are put together as listed in the following points.
Gap junction details in point form:
•Gap junctions are made up of clusters of closely packed connexons
•The connexons are hexameric: they consist of arrays of 6 connexin protein subunits
•Connexons pair up to form transmembrane channels
•The connexon hemichannel in one cell membrane docks with a connexon hemichannel in an adjacent cell
•About 20 connexin subunit isoforms exist in mammals
•A connexon may be made of the same (homohexameric) or different (heterohexameric) subunits
Figure 4.3. Ultrastructure of natural and purified gap junctions.
Gap Junctions and Their Regulation
In 1966, before gap junctions had been discovered, the existence of membrane junctions were hypothetically proposed to explain the flow of small molecules between certain cells. It was subsequently shown that many epithelial cells are physiologically- or electrically-coupled by the presence of unique structures called gap junctions. By injecting molecules into epithelial and other cells, it has been shown that small molecules including sugars, nucleotides, ions and signaling molecules can diffuse between cells through the connexons. The gap junctions of different cell types show different levels of permeability—different molecules can flow through at different rates depending on the cell type.
The movement of molecules through gap junctions was found to be determined by various characteristics such as the types of connexins that make up the connexons of the gap junction. The physiological state of the cell was also found to be important. However, there was a limit to the size of molecules that could enter and travel through connexons. Utrastructural studies (see above images) revealed that the actual channel size was limited and thus could exclude molecules based on their size. As shown in the following figure, microinjection experiments (e.g., flourescent dyes, labeled molecules, etc.) revealed that molecules larger than 5000 Daltons in molecular weight could not enter connexons while those that were smaller could (Figure 4.4).
Figure 4.4. Small molecules can pass through gap junctions while large ones cannot.
This was of great interest because it meant that large macromolecules (esp. proteins, DNA and RNA) could not move between cells via gap junctions. However, smaller molecules that are involved in intercellular communication could pass through as shown in the following figure (Figure 4.5). As will become clear when we discuss the roles of calcium ions, cyclic AMP and IP3 later in this book, the ability of small molecules to transfer between cells via gap junctions has important implications to cell function. What’s more by regulating the size of the connexon channels the intercellular movements of these small signaling molecules can also be controlled.
Figure 4.5. Cyclic AMP (cAMP), inositol 1,4,5 trisphosphate (IP3) and calcium ions (Ca2+) can pass through gap junctions.
Connexin Proteins Spontaneously Form Connexons
The connexin proteins were originally classified based on their molecular weights (e.g., Cx43 = 43kDa connexin protein). As more and more of them were identified, new ways of classifying them evolved but the old nomenclature is still used at times. As shown in the following experiment (Cx43 Experiment 1), pure liposomes are impermeable to the fluorescent dye Lucifer Yellow (Figure 4.6). When the purified gap junction protein Cx43 was added during the formation of the liposomes, the Lucifer Yellow was able to enter into the liposomes. Thus it is concluded the Cx43 can spontaneously form connexons which insert into the lipid bilayer allowing the dye to enter.
Figure 4.6. Experiments with the flourescent dye Lucifer Yellow revealed that gap junction proteins can spontaneously organize gap junctions to allow molecules to flow into cells. Note: liposomes are made up of a lipid bilayer but only a single lipid layer is shown for simplicity.
It was later shown that the flow of the dye through the connexon channels could be altered by phosphorylation of Cx43 (Figure 4.7). As detailed throughout this volume, protein kinases are enzymes that phosphorylate proteins. The simple addition of phosphate groups to proteins can significantly alter their function, so this is a critical event in cells. When mitogen activated protein kinase, MAPK, an important protein kinase involved in many signaling events is added to liposomes containing Cx43, the Lucifer Yellow dye molecules are unable to enter the liposomes. This suggests the MAPK phosphorylated the Cx43 making it unable to form permeable channels. When the MAPK was removed, the Cx43 was apparently dephosphorylated permitting the dye to pass through the functioning channels.
Figure 4.7. Experiments revealed that MAPK (mitogen activated protein kinase) can regulate the flow of molecules through gap junctions.Note: liposomes are made up of a lipid bilayer but only a single lipid layer is shown for simplicity.
In total, this work indicated that connexin proteins can spontaneously form connexons and that the functioning of those connexons can be regulated by the phosphorylation of the connexin protein.
Gap Junctions and Heart Function
Cardiac muscle is different from skeletal muscle because the contractile cells comprising it are connected electrically and not stimulated by nerves as is the case for skeletal muscle. Because cardiac muscle undergoes such strong regular contractions it has strong adhesion regions, called intercalated discs, that hold adjacent cardiac cells together. Adhesion components covered in the previous chapter (specialized adherens junctions, desmosomes) are also present. Cardiac muscle is comprised of a multitude of electrically insulated cells that can only communicate via gap junctions. Thus the number, size and localization of these gap junctions are critical to normal heart function. In keeping with this, disruption of cardiac gap junctions can lead to arrhythmias and other heart conditions.
At least five connexins (Cx43, Cx40, Cx45, Cx31.9 and Cx37) are expressed in the heart which is comprised of cardiac myocytes, vascular and interstitial cells, and other cell types (e.g., adipocytes; mesothelium). Different regions of the heart express different amounts and combinations of these connexins. Atrial myocytes express abundant amounts of Cx43 and Cx40 but only a very limited amount of Cx45. Connexin Cx43 is the primary ventricular gap junctional protein with only minor amounts of Cx45 and no detectable Cx40. Mouse knockouts for Cx43 show major heart malformations which lead to an early death.
Ventricular myocytes possess gap junctions that are among the largest of any mammalian tissue. In addition to the differences in connexin expression,
