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Chapter 4

Gap Junctions: Communication in the Heart and Glands

The normal functioning of the human body involves a vast diversity of communication events that are occurring simultaneously at the cell and tissue level in every organ of your body every millisecond of every day. As we will see in this chapter and those to follow, the disruption of any one of these signaling events can lead to a debilitating disease. After we summarize the types of intercellular signaling, we’ll detail the specialized intercellular structure called the gap junction that is essential to the functioning of a diversity of tissues and organs in the human body.

We can define three types of intercellular signaling in the human body (Figure 4.1):

•Endocrine. Cells in one part of the body send hormones via the bloodstream to influence other parts.

•Paracrine. Cells secrete substances that influence other cells around them.

•Autocrine. Cells secrete substances that influence themselves.

Figure 4.1. Types of signaling that occur between cells in the human body.

Thus these types of intercellular communications are defined by the distance between the signaling cell and its target cell. Intercellular signaling can also be classified based upon the way in which the signaling molecules from one cell type impact the target cells. These are called modes of intercellular communication. In spite of the complexity of the human body and the diversity of intercellular communications that occur, the modes of cellular communication can be classified into four major groups.

Four Types of Intercellular Communication:

•Diffusible Molecules (e.g., hormones, growth factors, neurotransmitters)

•Cellular Continuities (e.g., gap junctions)

•Cell Contact (e.g., adhesion molecule and its receptor)

•Mediated by the Extracellular Matrix (e.g., fibronectin, laminin).

Gap Junctions

Gap junctions are more accurately considered to be communicating junctions rather than cell adhesion junctions. But their structure likely results in both functions. Gap junctions have many important functions in cells. In the brain, gap junctions allow direct signaling between neurons, between glial cells and between neurons and glial cells. Gap junctions are also known to appear at specific times to mediate certain events. For example, gap junctions appear in the myometrium of the uterus during the later stages of pregnancy so that uterine contractions can be precisely controlled during childbirth. During embryonic development, gap junctions appear in developing muscle cells (myoblasts) to co-ordinate their fusion into future muscle fibers. This is covered in more detail in the chapter on biomembrane fusion.

Gap Junction Structure

The pictures below show the structure of gap junctions (Figure 4.2, 4.3). The first two panels in the image on the left of Figure 4.2, show how gap junctions appear in the transmission and scanning electron microscopes, respectively. The right-hand panel shows what purified gap junctional components look like.

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, heart gap junctions also exist in different numbers and patterns in different regions. Much remains to be learned about the functions of these different gap junctions.

Gap Junctions in Breast Development

Breast tissue is a secretory tissue designed for the storage and release of milk protein. Different types of connexin proteins are present in different regions of the developing breast indicating variations in the types of gap junctions that function in those regions (Figure 4.8). Focusing in on human breast duct in the following figure, note that Cx26 is expressed primarily in the duct luminal cells while Cx43 is localized in gap junctions in the contractile myoepithelial cells that regulate the release of the milk from the alveolus. Both of these gap junction proteins undergo developmental changes.

Figure 4.8. Connexin expression in mammary gland duct cells in mouse and human. ECM = extracellular matrix.

If Cx26 gene expression is knocked down in mice at puberty, alveolar development is impaired and lactation is prevented. If the Cx43 gene is knocked out the mice die at birth. A conditional knockout for Cx43 has not been generated. There is also evidence that in normal breast tissue both Cx26 and Cx43 have tumor suppressive roles.