Annual Review of Physiology - Volume 66, 2004

This article is mostly about the beginnings of the molecular biology of membranes, covering the decade 1964–1974. It is difficult to read (or write) this article because of a sense of déja vu. Most of the material in it is considered commonplace today, having been established experimentally since then. But at the time this work was begun, practically nothing was known about the molecular structure and the mechanisms of the functions of membranes. This situation existed because no membrane proteins of the kind I called integral had as yet been isolated in a pure state, and therefore none had had their amino acid sequence determined. The first integral membrane protein to be so characterized was human erythrocyte glycophorin, in 1978. It was the use of the thermodynamic reasoning that had been developed for the study of water-soluble proteins, together with the information from several key experiments carried out in a number of laboratories during the early decade, that led us to the fluid mosaic model of membrane structure in 1972. Without direct evidence to confirm the model in 1971–1972, my colleagues and I nevertheless had the confidence in it to pursue some of the consequences of the model for a new understanding of many membrane functions, which I present here in some detail. Finally, I discuss two recent high-resolution X-ray crystallographic studies of integral proteins to ask how well the structural and functional proposals that we derived from the fluid mosaic model fit these remarkably detailed X-ray results.

Heart failure remains a leading cause of hospital admissions and mortality in the elderly, and current interventional approaches often fail to treat the underlying cause of pathogenesis. Preservation of structure and function in the aging myocardium is most likely to be successful via ongoing cellular repair and replacement, as well as survival of existing cardiomyocytes that generate contractile force. Research has led to a paradigm shift driven by application of stem cells to generate cardiovascular cell lineages. Early controversial findings of pluripotent precursors adopting cardiac phenotypes are now widely accepted, and current debate centers upon the efficiency of progenitor cell incorporation into the myocardium. Much work remains to be done in determining the relevant progenitor cell population and optimizing conditions for efficient differentiation and integration. Significant implications exist for treatment of pathologically damaged or aging myocardium since future interventional approaches will capitalize upon the use of cardiac stem cells as therapeutic reagents.

In recent years there has been a rapid expansion in our understanding of the molecular biology that underpins human physiology. In the heart, elegant molecular pathways have been elucidated, and derangements in these pathways have been identified as factors in cardiac disease. However, as our understanding has grown, we have recognized that there exist only relatively crude tools to effect changes in molecular pathophysiology. The ultimate promise of gene therapy is to correct the molecular derangements that cause illness. To bring this promise to fruition in the clinical arena, many problems need to be solved, and chief among these remains reliable and robust delivery of genes to the target organ. To this end, viral vectors have been utilized with success more frequently than any other method of gene delivery. The use of these vectors in the heart has already offered promising novel benefit for human ischemic heart disease, and studies in animal models have given glimpses of hope that gene therapy may provide future therapeutic benefit in heart failure by improving cardiac function.

The developing distal lung epithelium displays an evolving liquid transport phenotype, reflecting a changing and dynamic balance between Cl⁻ ion secretion and Na⁺ ion absorption, which in turn reflects changing functional requirements. Thus in the fetus, Cl⁻-driven liquid secretion predominates throughout gestation and generates a distending pressure to stretch the lung and stimulate growth. Increasing Na⁺ absorptive capacity develops toward term, anticipating the switch to an absorptive phenotype at birth and beyond. There is some empirical evidence of ligand-gated regulation of Cl⁻ transport and of regulation via changes in the driving force for Cl⁻ secretion. Epinephrine, O2, glucocorticoid, and thyroid hormones interact to stimulate Na⁺ absorption by increasing Na⁺ pump activity and apical Na⁺ conductance (GNa⁺) to bring about the switch from net secretion to net absorption as lung liquid is cleared from the lung at birth. Postnatally, the lung lumen contains a small Cl⁻-based liquid secretion that generates a surface liquid layer, but the lung retains a large absorptive capacity to prevent alveolar flooding and clear edema fluid. This review explores the mechanisms underlying the functional development of the lung epithelium and draws upon evidence from classic integrative physiological studies combined with molecular physiology approaches.

Inward rectifiers are a class of K⁺ channels that can conduct much larger inward currents at membrane voltages negative to the K⁺ equilibrium potential than outward currents at voltages positive to it, even when K⁺ concentrations on both sides of the membrane are made equal. This conduction property, called inward rectification, enables inward rectifiers to perform many important physiological tasks. Rectification is not an inherent property of the channel protein itself, but reflects strong voltage dependence of channel block by intracellular cations such as Mg²⁺ and polyamines. This voltage dependence results primarily from the movement of K⁺ ions across the transmembrane electric field along the pore, which is energetically coupled to the blocker binding and unbinding. This mutual displacement mechanism between several K⁺ ions and a blocker explains the signature feature of inward rectifier K⁺ channels, namely, that at a given concentration of intracellular K⁺, their macroscopic conductance depends on the difference between membrane voltage and the K⁺ equilibrium potential rather than on membrane voltage itself.

Potassium (K⁺) channels exist in all three domains of organisms: eubacteria, archaebacteria, and eukaryotes. In higher animals, these membrane proteins participate in a multitude of critical physiological processes, including food and fluid intake, locomotion, stress response, and cognitive functions. Metabolic regulatory factors such as O2, CO2/pH, redox equivalents, glucose/ATP/ADP, hormones, eicosanoids, cell volume, and electrolytes regulate a diverse group of K⁺ channels to maintain homeostasis.

A vast number of proteins are involved in synaptic function. Many have been cloned and their functional role defined with varying degrees of success, but their number and complexity currently defy any molecular understanding of the physiology of synapses. A beacon of success in this medieval era of synaptic biology is an emerging understanding of the mechanisms underlying the activity of the neurotransmitter receptors for glutamate. Largely as a result of structural studies performed in the past three years we now have a mechanistic explanation for the activation of channel gating by agonists and partial agonists; the process of desensitization, and its block by allosteric modulators, is also mostly explained; and the basis of receptor subtype selectivity is emerging with clarity as more and more structures are solved. In the space of months we have gone from cartoons of postulated mechanisms to hard fact. It is anticipated that this level of understanding will emerge for other synaptic proteins in the coming decade.

Studies investigating the effects of temperature, food availability, or other physical factors on the physiology of marine animals have led to the development of biochemical indicators of growth rate, metabolic condition, and physiological stress. Measurements of metabolic enzyme activity and RNA/DNA have been especially valuable as indicators of condition in studies of marine invertebrates and fishes, groups for which accurate determination of field metabolic rates is difficult. Properly calibrated and applied, biochemical indicators have been successfully used in studies of rocky intertidal ecology, where two decades of experimentation have generated rigorous, testable models for determining the relative influences of biotic and abiotic factors on species distribution, abundance, and interaction. Biochemical indicators of condition and metabolic activity (metabolic enzymes, RNA/DNA) have been used to test nutrient-productivity models by demonstrating tight linkages between nearshore oceanographic processes (such as upwelling) and benthic rocky intertidal ecosystems. Indices of condition and heat stress (heat shock proteins, or Hsps) have begun to be used to test environmental stress models by comparing condition, activity, and Hsp expression of key rocky intertidal predator and prey species. Using biochemical indicators of condition and stress in natural systems holds great promise for understanding mechanisms by which organisms respond to rapid environmental change.

Whereas comparative physiology documents the range of physiological variation across a range of organisms, field physiology provides insight into the actual mechanisms an organism employs to maintain homeostasis in its everyday life. This requires an understanding of an organism's natural history and is prerequisite to developing hypotheses about physiological mechanisms. This review focuses on a few areas of field physiology that exemplify how the underlying physiology could not have been understood without appropriate field measurements. The examples we have chosen highlight the methods and inference afforded by an application of this physiological analysis to organismal function in nature, often in extreme environments. The specific areas examined are diving physiology, the thermal physiology of large endothermic fishes, reproductive physiology of air breathing vertebrates, and endocrine physiology of reproductive homeostasis. These areas form a bridge from physiological ecology to evolutionary ecology.

All our examples revolve around the central issue of physiological limits as they apply to organismal homeostasis. We view this theme as the cornerstone of physiological analysis and supply a number of paradigms on homeostasis that have been tested in the context of field physiology.

Although it is well established that during periods of torpor heterothermic mammals and birds can reduce metabolic rates (MR) substantially, the mechanisms causing the reduction of MR remain a controversial subject. The comparative analysis provided here suggests that MR reduction depends on patterns of torpor used, the state of torpor, and body mass. Daily heterotherms, which are species that enter daily torpor exclusively, appear to rely mostly on the fall of body temperature (Tb) for MR reduction, perhaps with the exception of very small species and at high torpor Tb, where some metabolic inhibition may be used. In contrast, hibernators (species capable of prolonged torpor bouts) rely extensively on metabolic inhibition, in addition to Tb effects, to reduce MR to a fraction of that observed in daily heterotherms. In small hibernators, metabolic inhibition and the large fall of Tb are employed to maximize energy conservation, whereas in large hibernators, metabolic inhibition appears to be employed to facilitate MR and Tb reduction at torpor onset. Over the ambient temperature (Ta) range where torpid heterotherms are thermo-conforming, the Tb-Ta differential is more or less constant despite a decline of MR with Ta; however, in thermo-regulating torpid individuals, the Tb-Ta differential is maintained by a proportional increase of MR as during normothermia, albeit at a lower Tb. Thermal conductance in most torpid thermo-regulating individuals is similar to that in normothermic individuals despite the substantially lower MR in the former. However, conductance is low when deeply torpid animals are thermo-conforming probably because of peripheral vasoconstriction.

Sleep and circadian rhythms are the primary determinants of arousal state, and torpor is the most extreme state change that occurs in mammals. The view that torpor is an evolutionary extension of sleep is supported by electrophysiological studies. However, comparisons of factors that influence the expression of sleep and torpor uncover significant differences. Deep sleep immediately following torpor suggests that torpor is functionally a period of sleep deprivation. Recent studies that employ post-torpor sleep deprivation, however, show that the post-torpor intense sleep is not homeostatically regulated, but might be a reflection of synaptic loss and replacement. The circadian system regulates sleep expression in euthermic mammals in such a way that would appear to preclude multiday bouts of torpor. Indeed, the circadian system is robust in animals that show shallow torpor, but its activity in hibernators is at least damped if not absent. There is good evidence from some species, however, that the circadian system plays important roles in the timing of bouts of torpor.

The past decade has witnessed a growing interest in estrogens and their activity in the central nervous system, which was originally believed to be restricted to the control of reproduction. It is now well accepted that estrogens modulate the activity of all types of neural cells through a multiplicity of mechanisms. Estrogens, by binding to two cognate receptors ERα and ERβ, may interact with selected promoters to initiate the synthesis of target proteins. Alternatively, the hormone receptor complex may interfere with intracellular signaling at both cytoplasmic and nuclear levels. The generation of cellular and animal models, combined with clinical and epidemiological studies, has allowed us to appreciate the neurotrophic and neuroprotective effects of estrogens. These findings are of major interest because estradiol might become an important therapeutic agent to maintain neural functions during aging and in selected neural diseases.

Nuclear receptors (also known as nuclear hormone receptors) are hormone-regulated transcription factors that control many important physiological and developmental processes in animals and humans. Defects in receptor function result in disease. The diverse biological roles of these receptors reflect their surprisingly versatile transcriptional properties, with many receptors possessing the ability to both repress and activate target gene expression. These bipolar transcriptional properties are mediated through the interactions of the receptors with two distinct classes of auxiliary proteins: corepressors and coactivators. This review focuses on how corepressors work together with nuclear receptors to repress gene transcription in the normal organism and on the aberrations in this process that lead to neoplasia and endocrine disorders. The actions of coactivators and the contributions of the same corepressors to the functions of nonreceptor transcription factors are also touched on.

Intestinal protein digestion generates a huge variety and quantity of short chain peptides that are absorbed into intestinal epithelial cells by the PEPT1 transporter in the apical membrane of enterocytes. PEPT1 operates as an electrogenic proton/peptide symporter with the ability to transport essentially every possible di- and tripeptide. Transport is enantio-selective and involves a variable proton-to-substrate stoichiometry for uptake of neutral and mono- or polyvalently charged peptides. Neither free amino acids nor peptides containing four or more amino acids are accepted as substrates. The structural similarity of a variety of drugs with the basic structure of di- or tripeptides explains the transport of aminocephalosporins and aminopenicillins, selected angiotensin-converting inhibitors, and amino acid–conjugated nucleoside-based antiviral agents by PEPT1. The high transport capacity of PEPT1 allows fast and efficient intestinal uptake of the drugs but also of amino acid nitrogen even in states of impaired mucosal functions. Transcriptional and post-transcriptional regulation of PEPT1 occurs in response to alterations in the nutritional status and in disease states, suggesting a prime role of this transporter in amino acid absorption.

Diarrheal diseases are among the most devastating illnesses globally, but the introduction of oral rehydration therapy has reduced mortality due to diarrhea from >5 million children, under the age of 5, in 1978 to 1.3 million in 2002. Variations of this simple therapy of salts and sugars are prevalent in traditional remedies in cultures world-wide, but only in the past four decades have the scientific bases for these remedies begun to be elucidated. This review aims to provide a broad understanding of the cellular basis of oral rehydration therapy. The features integral to the success of oral rehydration therapy are active glucose transport in the small intestine, commensal bacteria, and short-chain fatty acid transport in the colon. The review examines these processes and their regulation and considers new approaches that might supplement oral rehydration therapy in controlling diarrheal diseases.

Significant progress has been made in recent years toward understanding the mechanisms and regulation of intestinal absorption of water-soluble vitamins from the diet, especially those that are transported by a specialized carrier-mediated mechanism (i.e., ascorbic acid, biotin, folate, riboflavin, thiamin, and pyridoxine). The driving force involved in the uptake events and the molecular identity of the systems involved have been identified for a number of these vitamins. In addition, information about regulation of the uptake process of these micronutrients by intracellular and extracellular factors has been forthcoming. Furthermore, the 5′ regulatory region of the genes that encode a number of these transporters has been characterized, thus providing information about transcriptional regulation of the transport events. Also of interest is the identification of existence of carrier-mediated mechanisms in human colonocytes that are capable of absorbing some of the vitamins that are synthesized by normal microflora of the large intestine. Although the contribution of the latter source of vitamins toward overall host nutrition is not clear and requires further investigations, it is highly likely that it does contribute toward the cellular homeostasis of these vitamins in the localized colonocytes.

One of the central questions in neurobiology is how experience modifies neural function, and how changes in the nervous system permit an animal to adapt its behavior to a changing environment. Learning and adaptation to a host of different environmental stimuli exemplify processes we know must alter the nervous system because the behavioral output changes after experience. Alterations in behavior after exposure to addictive drugs are a striking example of chemical alterations of nervous system function producing long-lasting changes in behavior. The alterations produced in the central nervous system (CNS) by addictive drugs are of interest because of their relationship to human substance abuse but also because these CNS alterations produce dramatic, easily observed alterations in behavior in response to discrete stimuli. Considerable study has been given to behavioral and biochemical correlates of addiction over the past 50 or more years; however, our understanding of the cellular physiological responses of affected CNS neurons is in its infancy. This review focuses on alterations in cellular and synaptic physiology in the ventral tegmental area (VTA) in response to addictive drugs.

The intrinsic electrical properties of neurons are shaped in large part by the action of voltage-gated ion channels. Molecular cloning studies have revealed a large family of ion channel genes, many of which are expressed in mammalian brain. Much recent effort has focused on determining the contribution of the protein products of these genes to neuronal function. This requires knowledge of the abundance and distribution of the constituent subunits of the channels in specific mammalian central neurons. Here we review progress made in recent studies aimed at localizing specific ion channel subunits using in situ hybridization and immunohistochemistry. We then discuss the implications of these results in terms of neuronal physiology and neuronal mechanisms underlying the observed patterns of expression.

Given their prominent actin-rich subcellular specializations, it is no surprise that mechanosensitive hair cells of the inner ear exploit myosin molecules—the only known actin-dependent molecular motors—to carry out exotic but essential tasks. Recent experiments have confirmed that an unconventional myosin isozyme, myosin-1c, is a component of the hair cell's adaptation-motor complex. This complex carries out slow adaptation, provides tension to sensitize transduction channels, and may participate in assembly of the transduction apparatus. This review focuses on the detailed operation of the adaptation motor and the functional consequences of the incorporation of this specific myosin isozyme into the motor complex.