S0renPeter Olesen and Daniel B Timmermann

CONTENTS

13.1 Introduction 207

13.1.1 Ion Channels Are Pores through the Cell Membrane 208

13.1.2 Ion Currents Change the Electrical Membrane Potential 208

13.1.3 Gating of Ion Channels 209

13.1.4 Molecular Structures of Ion Channels 211

13.1.5 Ion Channels and Disease 211

13.1.6 Physiological and Pharmacological Modulation of Ion Channels 211

13.1.7 Drug Screening on Ion Channels 211

13.1.8 Structure of Voltage-Gated Ion Channels 212

13.2 Physiology and Pharmacology of Voltage-Gated Ion Channels—Potassium Channels 213

13.3 Voltage-Gated Calcium Channels 215

13.3.1 Structure and Molecular Biology 215

13.3.2 Physiological Roles of Voltage-Gated Calcium Channels 217

13.3.3 Pharmacology of Voltage-Gated Calcium Channels 217

13.3.3.1 Ca^-Family (L-Type Currents) 218

13.3.3.2 Ca^-Family (N-, P/Q-, and R-Type Current) 220

13.3.3.4 Auxiliary Subunits 220

13.4 Voltage-Gated Sodium Channels 221

13.4.1 Structure and Molecular Biology of Voltage-Gated Sodium Channels 221

13.4.2 Physiological Roles of Voltage-Gated Sodium Channels 221

13.4.3 Pharmacology of Voltage-Gated Sodium Channels 222

13.5 Chloride Channels 222

13.6 Ligand-Gated Ion Channels 223

Further Readings 223

13.1 INTRODUCTION

Ion channels form pores through the cell membrane, which are permeable to the small physiological ions Na+, K , Ca2+, and Cl-. The channels can open and close and thereby turn the flux of the charged ions through the cell membrane on and off. By this mechanism, the ion channels govern the fast electrical activity of the cells. Additionally, ion channels control Ca2+ influx and regulate responses as diverse as muscle contraction, neuronal signaling, hormone secretion, cell division, and gene expression. The opening of the channels is subject to regulation by physiological stimuli such as changes in membrane potential and ligand binding. Ion channels also lend themselves to pharmacological modulation and constitute important targets for drug treatment of diverse diseases including cardiac arrhythmia, arterial hypertension, diabetes, seizures, and anxiety.

13.1.1 Ion Channels Are Pores through the Cell Membrane

The cell membrane is impermeable to the small ions since they are charged. The ions polarize the water molecules around them and carry a shell of hydration water rendering them insoluble in the hydrophobic phospholipid membrane. Ion transport in and out of cells has to occur through specialized molecules, allowing the cells to compose a specific intracellular ion-milieu, which in many ways is different from the extracellular ion-milieu, e.g., there is more than a 10-fold gradient in the Na+- and K+-concentrations and a 10,000-fold gradient for Ca2+ across the cell membrane (Table 13.1).

The membrane proteins establishing these gradients are transporters such as the Na-K ATPase pumping three Na+ out and two K+ into the cell while consuming one ATP molecule. Other transporters are the Ca-ATPases pumping Ca2+ out of the cell or into the endoplasmic reticulum, and secondary active transporters such as the Na-Ca exchanger not using energy themselves but exploiting the gradients created by the ATPases. The transporters typically move 0.1-10 ions/ms each, they show saturation kinetics like enzymes, and they slowly build up the ion gradients.

The ion channels are different in many ways. They form water-filled pores through the cell membrane once they open, and permeation through the channels is only limited by diffusion. The transport is very fast, in the range of 104-105 ions/ms, and the opening of ion channels may change the membrane potential by 100 mV within less than 1 ms. Ion channels are thus in an ideal position to govern the fast electrical activity of cells.

TABLE 13.1

Typical Intra- and Extracellular Ion Concentrations and Corresponding Equilibrium Potentials

Intracellular Extracellular Equilibrium

Ion Concentration (mM) Concentration (mM) Potential (mV)

13.1.2 Ion Currents Change the Electrical Membrane Potential

In biological tissue electrical currents are conducted by the movement of ions. Bulk movements of ions in organs give rise to large currents resulting in voltage differences that can be measured on the body surface as was first done by Willem Einthoven in 1901 when he recorded human electrocardiograms. At the cellular level, the nature of excitable ion currents through the cell membrane was demonstrated by Hodgkin and Huxley in 1953 using a preparation of the squid axon. This giant nerve axon is about 1 mm in diameter, i.e., about 1000-fold thicker than human axons allowing electrodes to be positioned on either side of the membrane and the ion compositions on both sides to be controlled. Using this method the authors showed that selective movement of Na+ ions into the cell followed by an efflux of K+ ions is the basis for the electrical activity in nerve cells. The ions move passively across the cell membrane when the permeability increases, and the direction of the movement is determined by the combined chemical and electrical forces acting on them.

FIGURE 13.1 Cardiac action potential and time-course of selective Na+, Ca2+, and K+ currents.

These two forces are generated by the concentration gradient and by the electrical field generated by the membrane potential, respectively. The ion movement will stop once the two forces equal each other, which happens at the so-called equilibrium potential. This potential is determined by the ion distribution across the membrane, and for the typical intra- and extracellular ion concentrations the equilibrium potentials are shown in Table 13.1.

The effects on the membrane potential of activation on selective ion channels are shown in Figure 13.1. The cardiac action potential (AP) is initialized by opening of voltage-gated Na+ channels, and the influx of the positively charged Na2+ ions leads to a fast positive shift in the membrane potential (depolarization). Subsequently voltage-gated Ca2+ channels are opened and the influx of Ca2+ ions keeps the membrane potential depolarized. Fast K+ channels are activated early in the response and attenuate the depolarization, but the key role of the K+ currents is to terminate the AP after about 350 ms when numerous K+ channels open and the outflow of the positively charged K+ ions mediate the repolarization.

The consequence of the sequential opening of Na+-, Ca2+-, and K+-selective channels, is thus that the cell membrane potential will be pulled in the direction of the equilibrium potential for these ion species, i.e., about +70 mV for Na+, +120 mV for Ca2+, and -93 mV for K+ (Table 13.1). Often the cell does not fully reach the equilibrium potential as shown for the cardiac action potential, since several types of channels are usually open at the same time. Likewise the impact on the membrane potential of physiological or pharmacological ion channel block or activation depends on the presence of other simultaneous conductances and is not just linearly correlated to the number of ion channels being affected. Thus, it can be complicated to predict the functional effect of modulating ion channel function, and extensive target validation studies have to be conducted to establish the anticipated role of an ion channel subtype in an organ.

13.1.3 Gating of Ion Channels

While it was clear to Hodgkin and Huxley that a sequential increase in Na+ and K+ membrane conductances underlies the neuronal action potential, their method could not reveal the nature of the conductance pathway. This had to await another technological breakthrough. In 1976, Neher and Sakmann reported the opening and closing of single acetylcholine-gated ion channels in striated muscle using a method by which they electrically isolated a patch of membrane in situ with a glass pipette. The method was called patch-clamp with reference to the patch of tissue and the clamp of the transmembrane (TM) voltage used to generate the electrical driving force. Since then the method has been extensively used to describe the characteristics and function of ion channels in all cells. Initially endogenous currents in cells were measured, but following the cloning area the combination of this functional method and heterologous expression of cloned ion channels has been a strong combination in the target-driven drug discovery process.

111 Mil ill Illl 111 III_M , lilliil

Depolarization

Repolarization

Depolarization

Repolarization

Closed Open Inactivated

FIGURE 13.2 (A) Single channel recording of BK-type potassium channel. The baseline shown the closed state and the upward deflections are opening of single channels. The current through the channels is about 20 pA. (B) Opening, closing, and inactivation of ion channel. (Modified from Sanguinetti, M.C. and Tristani-Firouzi, M., Nature, 440, 463, 2006.)

Closed Open Inactivated

FIGURE 13.2 (A) Single channel recording of BK-type potassium channel. The baseline shown the closed state and the upward deflections are opening of single channels. The current through the channels is about 20 pA. (B) Opening, closing, and inactivation of ion channel. (Modified from Sanguinetti, M.C. and Tristani-Firouzi, M., Nature, 440, 463, 2006.)

Patch-clamp studies of single ion channels have shown that the duration of channel opening ranges from a few p,s to several hundred ms when exposed to a ligand or a voltage change (Figure 13.2). In the absence of stimulus they either open less frequently or stay closed. The opening and closing of ion channels is called gating, and at the single channel level it is described by the distribution of open- and closed-times. Currents through all ion channels in the cell membrane can also be measured by ripping a hole in the cell, which makes it possible to voltage-clamp the whole cell. This whole-cell current depicts the sum of hundreds of ion channels, and the kinetics of the current reflect the average open- or closed-times of the channels.

The gating is a dynamic process reflecting structural changes in the channel protein. The opening of a channel is preceded by conformational changes, and to the extent that these changes result in movement of charged segments of the molecule (voltage sensors), it can be followed by measurement of small gating currents. Once the channel goes into the open state, the electrical current carried by ions through the channel can be recorded with a resolution of about 1 pA (10-12 A). The activation of single channels is a discrete event, and as seen from the recordings in Figure 13.2 the ion channels are either fully closed or fully open. Thus, it is possible to follow the movements between the two conformational states with an amazing time- and current-resolution.

The various types of ion channels gate differently: some channels open only transiently whereas others stay open as long as the stimulus exists. Stimuli for ion channel activation are either (1) a change in the membrane potential, (2) a change in the concentration of extracellular ligands (neu-rotransmitters), (3) a change in the concentration of intracellular ligands (Ca2+, H+, cyclic nucleotides, or G-protein subunits), or (4) mechanical stimulation (e.g., stretch). Once the channels are exposed to the electrical or chemical stimuli they open or activate, and when the stimulus is removed, the channels close in an opposite process called deactivation. A number of channel types do however also close in the presence of the stimulus. It is a general physiological phenomenon that continued stimulation of a signal process results in a decreasing output. This functional closure of ion channels in the presence of stimulus is called inactivation and can occur either by parts of the channel protein plugging the open pore after a short delay, by collapse of the pore, or by decreased coupling between ligand-binding- and pore-domains (Figure 13.2B).

13.1.4 Molecular Structures of Ion Channels

Ion channels are present in all cells, and these naturally occurring channels have been extensively characterized with respect to gating kinetics, voltage- and ligand-sensitivity, pharmacology, and other parameters. In addition many ion channel types exhibit high affinity (pM or nM) to a number of toxins derived from scorpions, snakes, snails, or other animals, so toxins have been widely used to differentiate between the channels subtypes. The overall parameter used when describing an ion channel is its selectivity, i.e., whether it is selective to permeation of K+, Na+, Ca2+, or Cl-. Some channels are nonselective among cations. Since the selectivity is tightly coupled to the physiological function of the channels, this division is pragmatic and will be used in this chapter.

Following the sequencing of the human genome, 406 proteins with clear ion channel structure appeared. The characteristics of most of the cloned channels correspond well to the endogenous currents found in nerve, muscle, and other cells. The molecular constituents underlying other endogenous currents is however still debated, and these channels appear to be composed by several subunits from the same molecular family plus an additional number of accessory proteins. For voltage-gated channels the pore-forming subunits are denoted a-subunits, whereas the accessory subunits are called p, y or 5 subunits.

13.1.5 Ion Channels and Disease

The functional significance of specific ion channels in the body can be difficult to deduce from their molecular function, but it can be studied in organs or whole animals using pharmacological tools or selective toxins. Transgenic animals also provide valuable knowledge, but the most precise information about their role in humans has come from patients with diseases caused by dysfunctional ion channels. The diseases are typically caused by a point mutation in a single ion channel gene, and the diseases are jointly called channelopathies. The most frequent and well-known disease is cystic fibrosis, arising from a point mutation in the Cl- channel CFTR. In Northern Europe, 5% of the population is heterozygous for a mutation in the CFTR gene, and the prevalence of the disease is 0.5%e. Several types of cardiac arrhythmia (long and short QT syndromes, Brugada syndrome, and Andersen syndrome) are caused by mutations in cardiac K+, Na+, and Ca2+ channels. Mutations in neuronal and muscular ion channel subtypes cause epilepsy, ataxia, and myotonia. Luckily most of these are rare, but their study has given invaluable information about the role of the ion channels in health and disease.

13.1.6 Physiological and Pharmacological Modulation of Ion Channels

In addition to the main mechanisms for ion channel activation (voltage, ligands), the channels may also in some cases be modulated by small organic molecules. The ligand-gated ion channels exhibit an endogenous ligand-binding site, so compounds with similar functionalities can make potent drugs. The voltage-gated channels are not expected to naturally exhibit high-affinity binding sites, but may possess such as in the case of the dihydropyridine-binding site on the Ca2+ channel. Most drugs act as positive or negative modulators of the channel gating, but some may also just plug the pore as the local anesthetics blocking the neuronal Na+ channels or the neuromuscular blockers acting on the nicotinic channel in the neuromuscular junction.

13.1.7 Drug Screening on Ion Channels

The center-stage role of ion channels in many physiological responses has been stressed by functional studies in cells, organs, and animals, by the emerging channelopathies as well as by the successful use of ion channel modulating drugs. Current drugs only target a dozen of the known channel subtypes, while most of the other 400 types are currently all being investigated as potential drug targets in the pharmaceutical industry. Drug-discovery projects today depend strongly on large-scale blind-screening for finding new chemical lead molecules. The only high-throughput, high-quality technology to be used for screening on every ion channel subtype is the newly developed automated patch-clamp technique. With this method, parallel recordings are performed by a robot on 50-100 arrays of ion channel expressing cells positioned on silicon chips. Smaller throughputs can be obtained on arrays of 8-10 frog eggs expressing the desired ion channels, but the pharmacology of some channels may be different in this nonmammalian system. Channels giving rise to changes in the intracellular Ca2+ concentration can be screened using fluorescent Ca2+ dyes in a 384 well fluorescent reader (FLIPR) (see Chapter 12.3.2), which may also be useful for channels causing slow voltage changes. The use of other screens is typically limited to specific ion channels such as rubidium or thallium flux through K+ channels, or ligand binding to neurotransmitter-gated channels.

13.1.8 Structure of Voltage-Gated Ion Channels

The superfamily of voltage-gated ion channels encompasses more than 140 members and is one of the largest families of signaling proteins, following the G-protein-coupled receptors and protein kinases. The pore-forming a-subunits of voltage-gated ion channels are built upon common structural elements and come in four variations. The simplest version is composed of two TM segments connected by a membrane-reentrant pore-loop and having N- and C-termini on the inside (Figure 13.3). Four of such subunits form the channel. This architecture is typical for the so-called inward-rectifying K+ channels (Kir). It is found in a number of bacterial channels, suggesting it is the ancestor of the family. The second type is made by a concatenation of two such subunits, and the channel is formed by two double constructs. The third type is the 6-TM subunit, in which four extra membrane-spanning N-terminal domains including a voltage-sensor have been added to the basic 2-TM pore unit. Four of these 6-TM units form a channel. The group of 6-TM channels is rather large and includes the voltage-gated K+ channels (Kv), the calcium-activated K+ channels (KCa), the cyclic nucleotide-gated (CNG) channels, the hyperpolarization-gated channels (HCN), and the transient receptor potential (TRP) channels. Finally, the fourth channel structure type is made by concatenating four of the 6-TM subunits, making up a 24-TM subunit that forms the channel alone. This type is represented by the voltage-gated Na+ and Ca+ channels (Na and Ca). Within each of the four domains the six TM segments are denoted S1-S6.

Three different parts of the channels are responsible for the functions: ion permeation, pore gating, and regulation. The narrow part of the pore is called the selectivity filter, and this has been studied by high-resolution x-ray in crystallized K+ channels giving valuable insight into the selectivity mechanism (Figure 13.4). The residues in the pore loop line the selectivity filter and their carbonyl groups act as surrogate-water implying that the chemical energy of the dehydrated K+ ions entering

2-TM

4-TM

6-TM

24-TM

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