Introduction to the Human Cell. Danton PhD O'Day

      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.