Gap Junction Structure and Chemical Regulation: Direct Calmodulin Role in Cell-to-Cell Channel Gating

Gap Junction Structure and Chemical Regulation

Direct Calmodulin Role in Cell-to-Cell Channel Gating

Camillo Peracchia, M.D.

Professor Emeritus, Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, NY, United States

Table of Contents

Cover image

Title page

Copyright

Dedication

Foreword

Preface

Acknowledgments

Introduction

Chapter 1. Historical Background

1.1. Membrane Channels

1.2. Direct Cell-to-Cell Communication First Reported in Invertebrate Nervous System

1.3. Direct Electrical Communication Between Mammalian Cardiac Fibers

1.4. Direct Cell-to-Cell Communication in the Vertebrate Nervous System

1.5. Direct Cell-to-Cell Communication in Nonexcitable Cells

Chapter 2. Ultrastructure

2.1. Early Images of Gap Junctions in Invertebrate Excitable Cells

2.2. Early Images of Gap Junctions in Vertebrate Cells

2.3. The 60s Puzzle

2.4. Hexagonal Particle Arrays in Gap Junctions

2.5. Freeze-Fractured Gap Junctions

2.6. Early Models of Gap Junction Architecture

2.7. Correct Model of Gap Junction Architecture

2.8. Intracellular Gap Junctions? Do Gap Junctions Interact with Cytoplasmic Organelles?

2.9. How Do We Know That Gap Junctions Mediate Direct Cell-to-Cell Communication?

Chapter 3. Molecular Structure

3.1. Gap Junction Biochemistry

3.2. Early Crystallographic Studies

3.3. Amino Acid Sequencing of Connexins

3.4. Structural Studies Following the Identification of Connexin Sequences

Chapter 4. Chemical Gating—I. Role of Intracellular Calcium and Hydrogen Ions

4.1. Cytosolic Calcium and Gap Junction Channel Gating

4.2. Intracellular pH and Channel Gating

4.3. Does Ca2+i Mediate the Effect of Cytosolic Acidification on Channel Gating?

4.4. Does Ca2+i or pHi Play a Role in Channel Gating by Alkanols and Anesthetics?

4.5. What Causes [Ca2+]i to Increase With Cytosolic Acidification?

4.6. Does Ca2+i Have a Direct Effect on Channel Gating?

4.7. Is the Action of Ca2+ on Channel Gates Mediated by a Soluble Intermediate?

Chapter 5. Chemical Gating—II. Direct Calmodulin Role

5.1. CaM Inhibitors Prevent Cell-to-Cell Uncoupling

5.2. Inhibition of CaM Expression Prevents Cell-to-Cell Uncoupling

5.3. Testing the CaM Hypothesis by Expressing CaM-Mutants

5.4. Participation of CaM-Activated Enzymes

5.5. Colocalization of CaM and Connexins

5.6. Connexin Domains Potentially Relevant to Chemical Gating

5.7. Does Chemical Gating Require Cooperation Among Connexins?

5.8. Evidence of CaM Binding to Connexins and Connexin Peptides—Relevance to Channel Gating

5.9. Evidence That CaM Is Anchored to Connexins at Resting Intracellular Ca2+ Concentration

5.10. Hemichannels

5.11. Slow Vj Gate, Chemical Gate, and CaM

5.12. The Expression of the Cystic Fibrosis Transmembrane-Conductance Regulator Channel Increases Gj and Decreases Vj Sensitivity

Chapter 6. Chemical Gating—III. Models

6.1. Early Models

6.2. The Cork-Gating Model

6.3. Ca-CaM Locked-Gate—Irreversible Channel Gating

6.4. Reversible Ca-CaM-Cork Gating—Is There a Morphological Correlate of It?

6.5. Summary and Conclusion on Gating Mechanisms

Chapter 7. Connexin-Related Diseases

7.1. Nonsyndromic Diseases Caused by Connexin Mutations

7.2. Syndromic Diseases Caused by Connexin Mutations

Chapter 8. Future Perspectives

8.1. How to Balance Basic Versus Translational Research Funding?

8.2. How to Select Connexin Mutations for Understanding Domain Functions?

8.3. Role of Intracellular Connexins. Are There Intracellular Gap Junctions?

Further Reading

References

Index

Copyright

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Dedication

To the pillars of my life! My wonderful parents Luigi and Ida, and my adored wife and invaluable scientific collaborator Lillian Mae.

Cover: Freeze-fracture image of a gap junction between rat stomach epithelial cells uncoupled by treatment with 2,4-dinitrophenol (DNP). The gap junction is surmounted by the dumbbell image of a calmodulin (CaM) molecule.

Foreword

Dr. Camillo Peracchia, MD, is a Professor Emeritus in the Department of Pharmacology and Physiology at the University of Rochester Medical Center in Rochester, NY. He has published extensively on the subject of connexins (Cx) and gap junctions, and it was through these shared research interests that I had the opportunity to interact with him and expand my knowledge on the subject for my own research goals in understanding calcium signaling.

This book begins with historical puzzles about junctional structures of gap junctions that contain the intercellular channels for mediating the cell-to-cell transfer of small molecules (<1   kDa) between neighboring cells. He then introduces us to the scientists and the technological advances that gradually have unveiled the major mysteries of direct cell-to-cell communication. We now have a better understanding of how the regulation of Cx-mediated cell-to-extracellular and cell-to-cell communication plays essential roles in ionic selectivity, normal hearing, transparency of the lens in eyes, and synchronized contractions of the heart, as well as in various neurological disorders, and how they relate to several human diseases. Dr. Peracchia then shares his thoughts, insights, and the passion for knowledge that, for five decades, has driven his search for answers regarding the molecular mechanisms responsible for regulation of gap junctions.

After introducing early evidence for chemical gating, which dates as early as 1877 with Engelmann's healing over observation, the author provides a comprehensive analysis of research related to the roles of intracellular calcium and pH in the regulation of channel permeability. Through an array of meticulously executed experiments and step-by-step explanations, Dr. Peracchia presents his unique views on the discovery of calmodulin as a major direct regulator of the chemical gating of gap junction channels. He then provides an extensive and convincing review of recent research that has dissected the molecular mechanism of calmodulin regulation by means of a variety of structural and biochemical approaches.

As presented in this work, Dr. Peracchia's keen insights into gap junctions and their relationships to various diseases, his meticulously documented summary of major key concepts, and his deep personal wisdom regarding the challenges of research, all contribute to a text that is highly relevant to scientists, faculty members, students, trainees, and practitioners interested in cell biology. Dr. Peracchia's research, wisdom, and friendship have been especially important to me in my effort to understand the multifaceted roles of calmodulin and calcium signaling in various biological and pathological processes, including interactions with gap junctions. However, in a broader sense, Dr. Peracchia's legacy to science, and the world, will be the knowledge that he passes to the next generation of scientists, and the motivational influence he has provided me, and many others, to pursue our research goals with purpose, patience, and determination.

Jenny J. Yang, Ph.D

Regents' Professor of Biochemistry

Georgia State University

Preface

This book focuses on the structure and chemical gating of gap junctions channels, with special emphasis on the direct role of calmodulin (CaM) in channel permeability regulation. In the process, the narrative also follows the author's personal journey of over half a century in the field of direct cell-to-cell communication. The author feels fortunate of having started his work on gap junctions just 2   years after the major discovery of direct cell-to-cell communication in virtually all vertebrate and invertebrate organs because it has enabled him to witness first-hand the development of this major field of biomedical science from its very beginning. Indeed, the author's research on gap junctions began on April 19, 1966, the day he arrived in Boston from Milan (Italy) to join the laboratory of Dr. J. David Robertson (1923–1995) as fellow of Biophysics and Neuropathology at McLean Hospital (Belmont, MA), a branch of Harvard Medical School. A year later, his work continued in the Department of Anatomy of Duke University, and from October 1970 on in the Department of Physiology (presently Pharmacology and Physiology) of the University of Rochester, School of Medicine and Dentistry.

At first, the author's work focused on the ultrastructure of gap junctions in crayfish lateral giant axon and their changes with cell-to-cell electrical uncoupling and soon it expanded to the structure and function of vertebrate gap junctions. After about 15 years of ultrastructural work, in the early 80s the author began working on the electrophysiology of cell communication in Xenopus embryos and crayfish axons, and from late 90s on the molecular biology and gating mechanisms of gap junction channels made of wild-type or mutated connexins expressed in Xenopus' oocyte pairs.

In 1981, thanks to the excellent collaboration of Dr. Giovanni Bernardini and Lillian Mae Leverone Peracchia, the author's team was the first to report that CaM plays a major role in the regulation of gap junction channel gating (Peracchia, Bernardini, and Peracchia, 1981). Needless to say, we were quite excited, such that we submitted a paper to Nature. Unfortunately, the paper was rejected in part because our evidence for a CaM role in chemical gating conflicted with the favored view of the time that supported a major role of pHi, rather than Ca²+ i, in cell-to-cell channel gating. In retrospect, by rejecting our CaM idea, the major research team in the field did us a favor because had they accepted it and followed suit, they would have easily overshadowed our small team and received full credit for revealing the direct role of CaM in gap junction channel gating. So, for the following three-plus decades, we were by and large left alone in pursuing our studies on the direct CaM role in the gating mechanism.

Our original CaM findings, eventually published in Pflügers Archives (Peracchia, Bernardini, and Peracchia, 1983), prompted us to work full time on the electrophysiology, biophysics, and molecular biology of the chemical gating of gap junction channels. The discovery of connexin sequences in mid-1980s enabled us to test the effects of connexin mutations on the chemical regulation of gap junction channels expressed in Xenopus oocyte pairs. The direct role of CaM in chemical gating was then tested by a variety of approaches that included treatments with CaM inhibitors, inhibition of CaM expression, expression of CaM mutants, immunofluorescent colocalization of CaM and gap junctions, and binding of CaM to peptides mimicking connexin domains identified as potential CaM targets.

This book reports published findings but also proposes a number of hypotheses, speculations, suggestions, and ideas that could be tested experimentally. Of course, some of these may be proven wrong. Nonetheless, we feel that it is important to propose as many reasonable, testable, hypotheses as possible—this is the way science proceeds.

Acknowledgments

I owe debts of gratitude to James David Robertson who brought me to America over half a century ago and to my wife and invaluable scientific collaborator Lillian Mae Leverone Peracchia, as well as to many other collaborators for their great contribution to the effort of our research team, namely Brant S. Mittler, Angela F. Dulhunty, Maria E. Jaimovich, Giovanni Bernardini, R. Alberto Venosa, Peter Shrager, Stephen J. Girsch, Lili Shen, Patricia Donaldson, Stefano Giovannardi, Antonio Peres, Ahmed Lazrak, Ross G. Johnson and his team, Xiao Guang Wang, Li Quong Li, Feliksas F. Bukauskas, Anya (Ganna) Sotkis, Anthony Persechini, Kate C. Young, Joy T. Chen, Basilio (Bachi) Kotsias, Mohammad Salim, Katalin Török and her team, Nasr H. Anaizi, and many others who have contributed to our efforts over the years in various American Departments, namely: Department of Neuropathology (Harvard Medical School), Department of Anatomy (Duke University), Department of Physiology (University of Rochester, School of Medicine and Surgery), Department of Anatomy (Harvard Medical School), and Department of Pharmacology and Physiology (University of Rochester, School of Medicine and Surgery). I also express my sincere gratitude to the editors of Elsevier, in particular to Ms. Megan Ashdown, Mr. Debasish Gosh, Ms. Stacy Masucci and Ms. Mary Preap for their efficiency and professionalism.

Introduction

This book describes and discusses, without laying claims to completeness, major findings on the structure and chemical gating of direct cell-to-cell communication via gap junction channels. While knowledge of direct ionic communication between excitable cells of invertebrate nervous systems first surfaced in the early 20th century, before the mid-60s no one suspected that adjacent cells of virtually all tissues of vertebrates and invertebrates communicate directly with each other. The discovery of direct cell-to-cell communication in virtually all biological tissues opened a major chapter of biomedical science.

Following preface and introduction, eight chapters cover the major aspects of gap junction channel's structure and chemical gating. They include historical background, gap junction ultrastructure and architectural models, molecular structure, chemical gating, focused of the roles of cytosolic calcium, hydrogen ions, and calmodulin, models of chemical gating, connexin-related diseases, and future perspectives. This book will be of interest not only to established investigator who may need a comprehensive review of the field regarding gap junction structure and chemical gating but even more so to young investigators who may not be aware of how this important field of cell biology has developed.

Chapter 1

Historical Background

Abstract

This chapter reviews the major historical steps in the discovery of direct cell-to-cell communication. Evidence for electrical cell-to-cell communication in invertebrate nervous system, which first emerged in the early 1930s, was followed in the early 1950s by the discovery of membrane channels in excitable cells and their ability to mediate the rapid transmission of electrical impulses between invertebrate axons and between mammalian heart fibers. It took another decade, however, to learn that the direct cell-to-cell communication is also a property of virtually all cells, whether excitable or not. This major discovery, made accidentally in the mid-1960s, opened the door to a major field of physiology and pathophysiology.

Keywords

Cell theory; Crayfish; Earthworms; Electrical coupling; Electrical synapses; Electrotonic junctions; Giant axons; Insect salivary glands; Leech glia; Low-resistance junctions; Membrane channels; Metabolic coupling; Purkinje fibers; Septate axons

Our understanding of the way multicellular organisms operate most often has developed from the awareness of different biological activities long before the mechanisms and molecular components involved begun to be identified. In contrast, knowledge that the neighboring cells of most tissues directly communicate with each other developed more or less in an inverted order. In fact, before the mid-1960s, no one considered the possibility that neighboring cells of most tissues might exchange small cytosolic molecules. What for? Why would hepatocytes, for instance, need to exchange small molecules if they all perform the same function? Actually, knowledge of direct cell-to-cell communication via membrane channels permeable to small, charged, and neutral molecules came as a surprise. Indeed, this important function was discovered by accident, and its meaning in (electrically) nonexcitable cells puzzled the scientists who discovered it.

1.1. Membrane Channels

The cell theory, independently developed in the late 1830s by Matthias Jacob Schleiden (1804–1881) and Theodor Schwann (1810–1882), stated that tissues of plants and animals are made of independent units (cells) ¹,² (Schleiden, 1839; Schwann, 1839); rev. in (Turner, 1890). The concept of independent units obviously implied the existence of a wall-like structure (now known as the plasma membrane) functioning as a protective barrier to prevent the exchange of molecules with adjacent cells and the extracellular space. ³

However, while the cell theory did not consider the possible existence of membrane transport, toward the end of the 19th century, it became clear that certain molecules freely crossed the plasma membrane. The earliest studies on membrane transport did not consider the existence of channels but rather gave importance to the degree of lipid solubility of molecules (their oil–water partition coefficient) in determining their ability to diffuse in and out of cells. Plasma membranes were believed by the British physiologist Charles Ernest Overton (1865–1933) and others to be entirely made of lipids; rev. in (Kleinzeller, 1997). In the early 20th century, however, it became clear that this idea contradicted evidence for membrane transport of some lipid-insoluble molecules such as ions. This prompted the formulation of hypotheses suggesting the existence of hydrophilic pores or channels. Thereafter, membranes started being described as mosaics of "lipoid and sieve-like structures (Collander, 1937; Höber, 1936; Jacobs, 1924, 1935). Significantly, based on frictional properties and selectivity characteristics, in the mid-1930s membrane channels were already envisioned as narrow conduits with diameters similar to those of the permeant molecules (Jacobs, 1935) and composed of some fibrous protein" (Höber, 1936).

The earliest, definitive evidence of the existence of membrane channels came in the late 1940s and early 1950s through the work of Alan Lloyd Hodgkin (1914–1998) and coworkers (Hodgkin & Huxley, 1952a,b; Hodgkin, Huxley, & Katz, 1949; Hodgkin, Huxley, & Katz, 1952; Hodgkin & Katz, 1949). This major discovery was made possible by the earlier discovery of the giant squid axon (Young, 1938) and the inventions of microelectrodes by Ida Henrietta Hyde and the voltage-clamp technique (Cole, 1949); rev. in (Hille, 1992).

Several years later, evidence for the existence of channels capable of mediating direct cell-to-cell communications started emerging as well; rev. in (Peracchia, 1980; Loewenstein, 1975). These channels, now known as gap junction channels, are rather unconventional membrane channels not only because they span two membranes but also because, unlike other membrane channels, they are poorly selective, being permeable to both positively and negatively charged molecules, as well as to neutral molecules. Nonetheless, over the years, they have provided an excellent model system for studying the structure and function of membrane channels due to the fact that they are accessible to a large variety of technical approaches, spanning from crystallography to molecular biology, biochemistry, electrophysiology, biophysics, electron microscopy, and so on. For a review of 20th-century’s history of membrane channels see (Hille, 1992; Peracchia, 1994).

1.2. Direct Cell-to-Cell Communication First Reported in Invertebrate Nervous System

Evidence for the existence of membrane channels that mediate direct cell-to-cell communication in (electrically) nonexcitable cells emerged serendipitously in the mid-1960s (Loewenstein & Kanno, 1964; Kuffler & Potter, 1964; Kanno & Loewenstein, 1964). But, knowledge of direct ionic communication in some (electrically) excitable cells surfaced in the early 20th century from studies on invertebrate nervous systems (Stough, 1926, 1930).

1.2.1. Giant Axons

In the second half of the 19th century, zoologists discovered that certain annelids (earthworms), crustaceans (crayfish), and cephalopods (squid) display relatively large tube-like structures that extend from the rostral to the caudal end of the body. Most notable among them was the discovery of this type of structure in the squid (Williams, 1909)—a structure destined to become one of the most relevant research tools for studying the mechanism of action potential generation and the behavior of ion channels in general. Although most scientists of the time attributed different functions to these unusual structures, Franz von Leydig (1821–1908) was the first to suggest their nervous function (Leydig, 1864). His interpretation, however, was by and large ignored until the first half of the 20th century when John Zachary Young ⁴ (1907–97) unequivocally proved it right (Young, 1938).

1.2.1.1. Septa in Giant Axons—Evidence for Direct Axo-Axonal Electrical Communication

In the early 20th century, it became apparent that some of these giant tube-like structures (axons) were discontinuous. The first to realize it was George Edwin Johnson, who made this pivotal discovery in giant axons of crustaceans (Cambarus and Palaemonetes) (Johnson, 1924). Johnson's report clarified earlier data of Louis Boulé, who had shown by silver-impregnation neurofibril discontinuities that repeated at regular intervals along the length of the Lumbricus terrestris' giant axon (Boule, 1908)—a finding confirmed a couple of decades later (Smallwood & Holmes, 1927).

Two years after Johnson's report, similar structures segmenting earthworms' axons were named septa (Stough, 1926). In earthworms, there are three giant axons: a median and two lateral, all of them segmented. Howard B. Stough (1887–1976) described the septa as follows ⁵ : "…. there is a structure in the interior of the giant fiber which is most striking …. this is the cross-partition which completely divides the giant fibers segmentally (Stough, 1930). In his 1930s study, Stough found that when the median axon is cut, only the anterior portion of the worm contracts, extending posteriorly up to the cut area; in contrast, when the lateral axons are cut, leaving the median intact, posterior stimulation causes the posterior portion of the worm to contract up to the cut area. These data led Stough to conclude that: … the median giant fiber conducts antero-posteriorly and the lateral giant fibers conduct postero-anteriorly. The apparent polarization of these axons, however, was proven wrong 2   years later by John Carew Eccles (1903–97) and coworkers who recorded with an oscillograph impulses in the isolated nerve cord elicited by stroking with a feather either the head or the tail of the earthworm (Eccles, Granit, & Young, 1932). In the concluding remarks of their brief article, they stated that … the transverse membranes have no influence on the conduction of impulses, although the separation of the segments by means of these transverse membranes appears to be as complete as that existing at vertebrate synaptic junctions." A few years later, their findings were confirmed by several studies (Bullock, 1945; Rushton, 1945; Kao & Grundfest, 1957; Wilson, 1961).

Nonetheless, Stough deserves credit for being the first to demonstrate direct cell-to-cell electrical communication across morphologically definable septal barriers (Stough, 1926, 1930). In the absence of high-resolution images, however, it was not known then whether the current spread occurred via low-resistance membrane pathways or protoplasmic bridges. Three decades later, with the advent of electron microscopy, the detailed ultrastructure of the earthworms' septa was described as a very close apposition of plasma membranes with a total thickness of ∼200   Å (Hama, 1959) (see in the following).

Incidentally, by observing the apparent polarization of earthworms' median and lateral giant axons, Stough might have also inadvertently discovered the ability of injured cells to become electrically independent from each other (Stough, 1930). This property, now known as cell-to-cell uncoupling, is mediated by the gating mechanism of gap junction channels—most likely, in Stough's experiments the chemical gates of gap junction channels closer to the cut became activated by extracellular Ca²+ diffusing into the axoplasm.

In 1947 the nonpolarized transmission of electrical impulses across anatomically well-defined septa (Fig. 1) was also demonstrated in lateral giant axons of crayfish (Wiersma, 1947). This finding was later confirmed by studies that used microelectrodes for a more precise intracellular recording of electrical signals (Watanabe & Grundfest, 1961; Kao, 1960). By the late 50s and early 60s, evidence of electrical cell-to-cell coupling was also reported in several other invertebrates, such as the lobster's cardiac ganglion (Hagiwara & Bullock, 1957; Hagiwara, Watanabe, & Saito, 1959; Watanabe, 1958; Watanabe & Bullock, 1960) and muscle fibers (Reuben, 1960) and the segmental ganglia of the leech (Hagiwara & Morita, 1962; Eckert, 1963).

Figure 1 Crayfish ventral nerve cord. (A) Phase contrast image of cross-sectioned nerve cord showing median (M) and lateral (L) giant axons; the lateral axons are sectioned at the region (septum) where two axonal segments meet (see inset, white circle). (B) Electron micrograph showing a process of the caudal segment of a lateral giant axon penetrating through the septum to form gap junctions with the cranial segment; this junctional area is also seen in a phase contrast micrograph (inset a). At higher magnification (inset b), the cross-sectioned gap junction shows beaded profiles. The beads are junctional channels that link to each other across the extracellular gap. The channels' center-to-center spacing is ∼200   Å. The junction is flanked on both sides by rows of 500–800   Å vesicles.

1.2.1.2. Fenestrated Septa in Crayfish Axons

Incidentally, in addition to the septa, crayfish axons display other unusual structures that, although likely to be present also in vertebrate axons, are more easily recognizable in crayfish (Figs. 2 and 3). Phase-contrast images of longitudinally sectioned crayfish nerves, fixed with glutaraldehyde-H2O2 (Peracchia & Mittler, 1972), display regularly spaced striations crossing the axoplasm every ∼2   μM (Peracchia, 1970; Fig. 2A). Electron microscope images revealed that each striation is made of two cross-sectioned membranes separated by 150–400   Å spacing (Fig. 2B–E). The two membranes are the profiles of "fenestrated septa" (Peracchia, 1970) or transverse cisternae (Fig. 2C–E). The membranes of the fenestrated septa frequently join, forming 0.1–0.2   μM pores each occupied by a single microtubule (Fig. 2C). Neighboring fenestrated septa are joined by longitudinal membranous tubules (Fig. 2B, D and E). In cross-sectioned axons the septal membranes, now seen in