cu .V. ntroduction II. T he Pharmacology of the Macroscopic Skeletal Muscle Cl Conductance gCl III. N ew Molecules Targeting ClC-1 Identified Using Heterologous Expression IV. M echanism of Block of Muscle Type CLC Channels by Clofibric Acid Derivatives V. T he Binding Site for 9-AC and CPA on ClC-1 and ClC-0 and Use of CPA as a Tool to Explore the Mechanisms of Gating of ClC-0 VI. P harmacology of CLC-K Channels VII. O ther CLC Channels and Other Blockers V III. P otential of Having Blockers of ClC-2, ClC-3, ClC-5, ClC-7: Outlook R eferences
and l ri he Channels B. T he Transporters IV. A Multi‐Ion Pore V. C hannels and Transporters: Where Is the Difference? VI. F unction of the CLC Transporters VII. S peculative Modeling V III. C LC Channel Gating A. V oltage, Cl and Hþ Gating of a CLC Channel B. M olecular Identity of the Fast Gate C. T he Slow Gate D. F ast and Slow Gating in Other CLC Channels IX. T he Role of the C‐Terminal Domain of CLC Channels: Recent Developments X. C onclusions and Outlook R eferences
CLC genes are expressed in species from bacteria to human and encode Cl(-)-channels or Cl(-)/H(+)-exchangers. CLC proteins assemble to dimers, with each monomer containing an ion translocation pathway. Some mammalian isoforms need essential beta -subunits (barttin and Ostm1). Crystal structures of bacterial CLC Cl(-)/H(+)-exchangers, combined with transport analysis of mammalian and bacterial CLCs, yielded surprising insights into their structure and function. The large cytosolic carboxy-termini of eukaryotic CLCs contain CBS domains, which may modulate transport activity. Some of these have been crystallized. Mammals express nine CLC isoforms that differ in tissue distribution and subcellular localization. Some of these are plasma membrane Cl(-) channels, which play important roles in transepithelial transport and in dampening muscle excitability. Other CLC proteins localize mainly to the endosomal-lysosomal system where they may facilitate luminal acidification or regulate luminal chloride concentration. All vesicular CLCs may be Cl(-)/H(+)-exchangers, as shown for the endosomal ClC-4 and -5 proteins. Human diseases and knockout mouse models have yielded important insights into their physiology and pathology. Phenotypes and diseases include myotonia, renal salt wasting, kidney stones, deafness, blindness, male infertility, leukodystrophy, osteopetrosis, lysosomal storage disease and defective endocytosis, demonstrating the broad physiological role of CLC-mediated anion transport.
A major function of transporting epithelia is vectorial ion movement. This is accomplished by ion channels, pumps, antiporters, and symporters. The activity of these transport proteins is regulated by diverse signaling transduction and membrane trafficking events, many of which involve the actin cytoskeleton. This chapter will focus on the role of actin microfilaments and associated proteins in regulation of ion transport. The apical Na+H+ antiporter NHE3 will be examined in detail in this context due to its general biological importance as well as recent advances that have helped to elucidate the mechanisms of regulation of this protein. Appreciation of the mechanisms of NHE3 regulation should serve as a framework with which to understand cytoskeletal regulation of other transport proteins as well.
At least 18 different cytoskeletal proteins have been shown to interact in vitro with, and in some cases are regulated by, specific membrane lipids, mostly phosphoinositides, whose synthesis is themselves regulated by extracellular signals. These lipid interactions are mediated by structurally diverse specific binding sites. Lipid interactions can serve to target proteins, such as spectrin, myosin X, dynamin, or annexin 2, to the plasma membrane or inactivate processes counteracting actin polymerization and stimulate proteins, such as Wiskott–Aldrich syndrome protein (WASP), involved in nucleating actin polymerization. In other cases (ezrin, talin), phosphoinositides serve to activate proteins involved in actin‐membrane linkage. Such interactions may occur highly localized in lipid rafts.
During embryogenesis, the tissues of the developing embryo must be bent, tugged, and sculpted in a series of morphogenetic episodes that flow from one to another to generate the final shape of the fetus. The mechanisms underlying these events share startling similarities across a great variety of animals. A primary component of morphogenesis is the actin cytoskeleton, which drives all of the cell shape changes, the tissue bending, and the fusion events that characterize embryogenesis. In this chapter we explore how regulation of the actin cytoskeleton contributes to morphogenetic episodes in worm, fly, fish, mouse, and chick embryos. In particular, we focus on how each of the different structures formed by actin function in morphogenesis and consider how recent research has expanded our knowledge of this area.
Villin is an epithelial cell‐specific protein that belongs to a family of actin‐binding proteins that display structural and functional homology and include among other proteins, gelsolin, severin, fragmin, adseverin/scinderin, supervillin, and protovillin. Villin is unique among these proteins in that it can sever, cap, nucleate, and crosslink actin filaments. The actin modifying functions of villin are regulated by calcium, phosphoinositides, and tyrosine phosphorylation of the villin protein. Villin also self‐associates and this property of villin is likewise determined by its ligand‐binding properties. Overexpression of villin regulates actin redistribution, cell morphology, cell migration, and cell death. Studies done with the villin knockout mice substantiate the significance of villin in actin reorganization and maintenance of epithelial cell plasticity. In this chapter I discuss how the ligand‐binding properties of villin are mechanistically important to its functions in cell migration and cell death.
The positioning of the endomembrane system (biosynthetic/secretory and endocytic pathways) and the constant movement of constituents between these compartments requires the involvement of the cytoskeleton and cytoskeletal‐based motors. Whereas much is known about the roles of the microtubule (MT) cytoskeleton in these events, considerably less is known about the roles of the actin cytoskeleton. Recent work has shown that the Golgi complex is linked to an actin‐based network that is important for Golgi morphology and the trafficking of Golgi‐derived membranes. Moreover, many of the cellular players used to transport cargo near the actin‐rich network subjacent to the plasma membrane (PM) may function near the Golgi.
Spectrin and ankyrin are major components of a plasma membrane‐associated scaffold known as the spectrin cytoskeleton. Spectrin is thought to cross‐link actin filaments in a two‐dimensional submembrane network and ankyrin links the network to a number of integral membrane activities, including the sodium pump, voltage‐dependent sodium channels, and L1‐family cell adhesion molecules. Genetic studies have established that the spectrin cytoskeleton is required for the normal accumulation of these integral proteins at their normal sites of function. Defects in spectrin and ankyrin have been implicated in human disease, and studies in model genetic organisms are making it possible to elucidate the mechanisms behind their effects. Cell culture studies have also shed new light on the processes that govern formation of polarized membrane domains. This chapter surveys the results and their implications for the assembly and function of the spectrin cytoskeleton.
Intermediate filaments (IFs) are cytoskeletal structures that maintain cell and tissue integrity. In addition, they have been implicated in a number of other functions, including maintenance of cell shape, subcellular organelle positioning, support to cell migration, radial growth of axons, and scaffolding of signaling molecules. Many of the recognized tasks are affected by phosphorylation, which is the key mechanism to regulate both the organization of IFs and the association of IFs with interacting molecules. Another important function of IF phosphorylation is the disassembly of cytoplasmic and karyoskeletal IF networks during mitosis. An intriguing feature of IFs is the tripartite structure of their building blocks, with a highly conserved central rod domain, which is flanked by variable length N‐ and C‐terminal regions. While the rod domain is crucial for the assembly of IFs, a common feature of the distal domains is that they represent the main targets of phosphorylation. Since phosphate targeting seems to have domain‐specific functions, this chapter aims at summarizing the different roles of N‐ and C‐terminal phosphorylation of IFs.
