Introduction

Actions speak louder than words. The molecular roots of our actions and the thoughts and feelings that drive us to act are ion channels, proteins that form macromolecular pores in cell membranes. These transmembrane proteins generate and propagate the electrical signals that allow us to sense our surroundings, process information, make decisions, and move.

Ion channel proteins act as gates that span the lipid bilayer that surrounds all cells where they open and close to allow the flow of ions down their electrochemical gradients (Fig. 1). The ion flux through a channel pore can be extremely high, «106 ions per second [1]. Because of their central role in the function of the excitable tissues such as heart, brain, muscles, and nervous system, investigators have long sought to understand ion channel properties from a molecular perspective. Decades of biophysical measurements and functional studies have been devoted to understanding ion channel function [1]. Yet, the very nature of these molecules—transmembrane proteins that are difficult to obtain in the large quantities and high purity necessary for structural investigation—has impeded attempts to obtain the most essential information for understanding their functions, a three-dimensional description of their molecular architectures at high resolution. In the past 5 years, the once impregnable barrier separating biophysicists and neurosci-entists from this essential information has been breached.

The first high-resolution structures of ion channels and ion-channel-associated proteins are providing the substrates for sophisticated tests of the mechanisms of channel gating and permeation. This chapter touches briefly on these pioneering studies and the questions they raise.

Ion channels perform two basic functions. They open and close to control the passage of ions across the cell membrane (see Chapter 36) and they sense and respond to signals that drive them between open and closed states (see Chapters 35 and 37 to 40). The response times of channels to these inputs can be very fast, on the order of tens of microseconds to a few milliseconds [1]. Different classes of ion channels have been designed by nature to respond to the three types of signals one can imagine sensing in a membrane environment: extracellular signals such as neurotransmitters (e.g., acetylcholine and glutamate receptors; see Chapters 37 and 38), transmembrane voltage changes (typified by voltage-sensitive cation channels; see Chapter 35), and intracellular signals such as calcium and cyclic nucleotides (see Chapters 39 and 40). While channels are generally classified based on the primary signal that opens them, many channels serve as integrators and respond to some combination of signals.

The pore-forming domains of most ion channels are multimeric assemblies possessing cyclic symmetry in a general architecture known as barrel-stave (Fig. 2). A fixed number of subunits assemble around the axis of the ion conduction pore.

Extracellular M^^HI

(voltage change,tigand binding, J

second messenger) %

Cytoplasm

Figure 1 Schematic of ion channel function as viewed from the plane of the membrane. Three subunits of an ion channel are shown in magenta; ions are shown as green spheres. Upon activation by a stimulus, such as a transmembrane voltage change or ligand binding, the channel undergoes a conformational change that opens a pore formed by the protein. Ions flow through the open pore in a direction that is determined by the electrochemical gradient.

Figure 2 General architecture of ion channels. Parts (a) and (b) show the barrel-stave characteristics of the voltage-gated cation channel family and the nicotinic acetylcholine receptor family. In each of these, the channel subunits are arranged around the pore through which the ions flow. Part (c) shows the general architecture of voltage-gated chloride channels. These channels are dimers in which each subunit makes its own pore. (Adapted from Jentsch, T., Nature, 415, 276-277, 2002.)

Figure 2 General architecture of ion channels. Parts (a) and (b) show the barrel-stave characteristics of the voltage-gated cation channel family and the nicotinic acetylcholine receptor family. In each of these, the channel subunits are arranged around the pore through which the ions flow. Part (c) shows the general architecture of voltage-gated chloride channels. These channels are dimers in which each subunit makes its own pore. (Adapted from Jentsch, T., Nature, 415, 276-277, 2002.)

The number of subunits is roughly related to the size and selectivity characteristics of the channel. For example, the most selective channels, such as voltage-gated sodium channels and voltage-gated potassium channels, are tetramers in which four identical or highly homologous subunits are arranged around the pore. Pentameric channels such as the nicotinic acetylcholine receptor (nAChR) have larger pores and generally discriminate between positive and negative ions but not among ions within these general classes. Hexameric channels such as gap junctions allow ions and small solutes to pass [2]. The barrel-stave channel arrangement has been a boon to structure-function studies, as the channel symmetry imposes strong constraints on the likely location of amino acids close to the pore. Nature, however, does not always follow this plan when constructing ion channels. Voltage-gated chloride channels have two pores that are formed from a dimer of subunits in which each subunit makes its own ion passageway (Fig. 2c) [3].

Furthermore, good overexpression systems for producing eukaryotic membrane proteins in the quantities required for high-resolution studies are not currently available. Solving this technical problem is one of the major requirements for routine high-resolution investigation of membrane protein structure.

Electron microscopy studies have proven particularly useful in obtaining low- to medium-resolution descriptions of eukaryotic ion channels and the conformational changes that accompany ion channel opening. Studies of ion channels found in high abundance in the electric organs of electric rays and electric eels, such as the nicotinic acetylcholine receptor and the voltage-gated sodium channel [4,5], reveal the general cyclic symmetric architecture of both of these channels (Fig. 3). While difficult, these studies require much less protein than other structural methods, and information can be obtained from two-dimensional crystals, tubular membrane crystals, and even single particles.

Studies of Full-Length Ion Channels

X-ray crystallographic and nuclear magnetic resonance experiments are the most powerful tools for obtaining information about the atomic structure of macromolecules. Unfortunately, it is still extremely difficult to use these methods to study membrane proteins such as ion channels. Ion channels have domains that reside in the hydrophobic environment of the cell membrane as well as domains that reside in the aqueous intra- and extracellular spaces. To keep the transmembrane domains soluble upon removal from the cell membrane, reagents such as detergents or lipids must be used in the purification and handling of full-length channels. The search for the precise detergent or lipid that will work for a given channel complicates purification attempts as well as the search for conditions that produce diffraction-quality protein crystals, the necessary prerequisite for any X-ray crystal-lographic study. The large size of most ion channel proteins places them outside what is currently possible with the most sophisticated nuclear magnetic resonance (NMR) methods.

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