STRUCTURAL FEATURES AND FUNCTIONAL MECHANISM OF VOLTAGE-GATED ION CHANNELS ADINA-LUMINIŢA MILAC * Laboratory of Cell Biology, CCR, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4258 USA (Received October 18, 2010) Voltage-gated ion channels are an important class of drug targets not only for neuromuscular diseases, but also for cancer and autoimmune disorders. Unfortunately, the structure-based drug design is hampered by limited structural information and insufficient understanding of the activation mechanism. This review gives a state-of-art overview of the current knowledge on the structure and activation mechanisms of voltage-gated ion channels and of the (still) open questions in the field. Key words: ion channels, voltage sensing, structural biology. INTRODUCTION Nervous impulses are transmitted along axons at velocities up to 120 m/s. To perform this work, the cell membrane quickly and accurately changes its permeability to various ions (1). There is an equilibrium voltage different than 0 V between the intra-cellular and extra-cellular medium, for each type of ion. There are four main ions involved in this process: Sodium (Na), Potassium (K), Chlorine (Cl) and Calcium (Ca). Each of them has different permitivity (equivalent to conductance) through soma membrane and different equilibrium potential. This leads to the existence of a global equilibrium potential called Resting Potential. In most neurons the resting potential has a negative value of about -70mV, which by convention means that there is excess negative charge in the intra-cellular medium. The permeability to different ions is mediated by selective ion channels forming transmembrane pores. The channels are opened and closed by different stimuli such as neurotransmitters, membrane stretch, temperature, and transmembrane voltage. Depending on the design of the pore, they are selective for specific ions. The permeability changes necessary for transmitting the nervous impulse are caused by the opening and closing of different voltage-gated ion channels that respond to changes in the membrane potential (2). * Corresponding author (E-mail: milac.adina@gmail.com) ROM. J. BIOCHEM., 47, 2, 193 201 (2010)
194 Adina-Luminiţa Milac 2 Voltage-gated ion channels are key players of the nervous and muscle system function, forming the basis for conduction of nervous impulses, muscular contraction, and synaptic transmission. These channels (together with closely related voltage-insensitive channels) form the third largest superfamily of signal-transduction proteins, only outnumbered by G protein-coupled receptors and protein kinases, comprising 143 members in the human genome (3). In this family classical voltage-gated Na, Ca, and K channels are found together with for instance Ca 2+ -activated, cyclic nucleotide-gated, and hyperpolarization-activated channels. The function of ion channels can have a strong impact on cellular function and cellular signaling. Ion channels are required in multiple normal physiological processes, such as muscle contraction and neuronal signal transmission. Thus, dysfunctional channels cause disease (4) and a large number of medical drugs, as well as animal and plant toxins, use them as target. Historically, the role of ion channels was most obvious in the membrane of electrically excitable cells, such as the neuron, the cardiac myocyte, and the skeletal muscle fiber. Consequently, a number of drugs able to modulate cell excitability by acting on voltage-gated or neurotransmitter-gated ion channels in these tissues have reached blockbuster status in the pharmaceutical industry, generating large profits. Examples are the antiepileptic drugs (AEDs), which include blockers of voltage-gated sodium and calcium channels, agonists of GABAA receptors, and, more recently, openers of potassium channels and antagonists of AMPA and NMDA glutamate receptors. But defects in ion channel function can also cause disorders outside the neuromuscular spectrum. For example, it has been shown that the voltage-gated Kv1.3 channel and the intermediate conductance calcium-activated potassium channel, IKCa1, play important roles in controlling T-cell activation (5, 6). Selective blockers of Kv1.3 and IKCa1 channels could serve as effective immunosuppressants (7). In addition to the potassium channels expressed in immune cells, potassium channels from the ether-a-go-go family (eag, erg, and elk) have been suggested to play important roles in controlling cancer cell proliferation (8, 9). In general, most medical drugs in clinical use targeting ion channels block the ion-conducting pore. However, the voltage sensing machinery is an alternative and suitable target for medical drugs that could tune channel activity and thereby also neuronal and cardiac excitability. While the ion conducting pore is known at atomic resolution for several ion channels (10 14), the structure of voltage sensing domain and the mechanism by which the channels sense transmembrane voltage is known in less detail (15, 16). This represents the greatest impediment to structurebased drug design and discovery of effective drugs with increased selectivity, therefore current research is focused on elucidating functional mechanisms of voltage-gated ion channels.
3 Ion Channels Voltage-Sensing Mechanism 195 STRUCTURE OF VOLTAGE-GATED CHANNELS Voltage-gated ion channels are composed of four subunits (or four linked domains as in Na and Ca channels) symmetrically arranged around a central ion-conducting pore. Na and Ca channels are single polypeptides, organized in four linked domains, while K channels are formed by four separate subunits. These subunits consist of assemblies of transmembrane α helices. For K channels they are essentially of two types: two transmembrane helices (2TM) or six (6TM). The α helices are labeled M1 and M2 in the 2TM channels, and S1 to S6 in the 6TM channels. M1 and M2 are homologous to S5 and S6. Each subunit of the Kv channel contains six transmembrane segments named S1 S6 and has a modular organization: the pore (ion-conducting) domain (S5 S6) in which the linker between S5 and S6 forms a selectivity filter and the voltage-sensor domain (VSD; S1 S4). At the intracellular half of the pore, the four S6s form an inner vestibule, at the internal end, limited by a narrow passage (a gate) that can open up when the channel is activated. Via the S4 S5 linker helix, the gate is coupled to the four VSDs located outside the pore-forming unit. This coupling enables channel opening and closing upon changes in the membrane electric field. Most channels open at positive voltages, but there are also channels that open at negative voltages like the hyperpolarization-activated HCN channels. STRUCTURE OF VOLTAGE-SENSING DOMAIN When 6TM voltage-gated channels were first cloned, the fourth putative membrane-spanning segment S4 stood out because it contains many basic Arg or Lys residues that are capable of carrying a positive charge (17 21). The S4 segment is always found together with three other transmembrane segments (S1 S3) that contain negatively charged acidic residues, and the four segments together collectively form an S1 S4 voltage-sensing domain. The concept that S1 S4 is a domain in the formal sense grew out of comparisons of Kv channels and other simpler K + channels that lack the S1 S4 region (22), experiments identifying important acidic residues in S2 and S3 (23), and the discovery that tarantula toxins can bind to S1 S4 domains in different types of voltage-activated ion channels (24). That S1 S4 voltage sensors are functionally independent protein domains was demonstrated by Lu and colleagues when they found that the S1 S4 region of a Kv channel could endow the KcsA K + channel with strong voltage-sensitivity (25, 26). Bacteria can synthesize, properly fold and insert isolated S1 S4 voltage sensing domains into their membranes (27, 28), further supporting the idea that S1 S4 is an independent domain.
196 Adina-Luminiţa Milac 4 For many years, the lack of three dimensional structures of voltage sensors was a barrier to understanding the mechanism of voltage sensing. At present, four X-ray structures have been solved which contain S1 S4 voltage sensing domains (28 30). As will be discussed below, these structures are probably most relevant for thinking about the activated state of voltage sensors that exist at depolarized membrane voltages. The first two structures reported in 2003 by MacKinnon and his colleagues were of the isolated S1 S4 domain and full-length channel of KvAP (28), an archaebacterial Kv channel from Aeropyrum pernix. The structure of the isolated S1 S4 domain comprises five helices, with S1, S2 and S4 forming long uninterrupted helices and S3 containing two shorter helices (Fig. 1), consistent with helix scanning studies in eukaryotic Kv channels (31 34). Because this domain was crystallized without the pore domain, it was not obvious how it is oriented with respect to the pore or the surrounding membrane. The structure of the full-length KvAP channel did not answer this question because the voltage sensing domains in this structure are distorted (28). Fig. 1. Structure of natural voltage gated ion channels. The X-ray structure of the eukaryotic Kv1.2 channel provided the first clear perspective of S1 S4 domains in a full-length channel and revealed how they are oriented with respect to the central pore domain and the surrounding lipid membrane (35). Even though the quality of electron density within the S1 S4 domain is relatively weak (mean B factor = 162 Å 2 ), leaving many residues unresolved, the S1 through S4 helices clearly adopt transmembrane orientations and appear loosely attached to the central pore domain. The most recent X-ray structure of the Kv1.2 channel containing the paddle motif from the Kv2.1 channel (paddle-chimera channel) (30) currently provides the clearest picture of voltage sensor structure in a full-length channel (Fig. 2). The close apposition of the S3b S4 helices are similar to what is seen for the paddle motif in the KvAP structures, S1 and S4 are positioned adjacent to the pore domain and both S2 and S3 are
5 Ion Channels Voltage-Sensing Mechanism 197 VSD PD Fig. 2. The paddle-chimaera channel, in which the voltage-sensor paddle has been transferred from Kv2.1 to Kv1.2. Voltage-Sensing Domain (VSD) and Pore Domain (PD) are indicated. positioned peripherally. Electron densities for many lipid molecules are also present in the maps for the paddle-chimera channel, revealing that lipids interact intimately with the channel. Lipids are present between the S1 S4 voltage-sensing domain and the pore domain, between adjacent S1 S4 domains and even intercalating between helices. Superposition of the structures of the paddle-chimera and the isolated S1 S4 domain of KvAP reveals that the structures of voltage sensors are well conserved from archaebacteria to humans VOLTAGE SENSOR MOTION AND VOLTAGE SENSING MECHANISM Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how transitions occur in membrane proteins not to mention numerous applications in drug design. The first model to describe the S4 movement was the helical screw model (36, 37). As depicted in Fig. 3A, the positive charges are lined up in a spiral around
198 Adina-Luminiţa Milac 6 S4. They are supposed to pair with conserved negative counter charges in S2 and S3. Upon activation, S4 moves to new stable positions by rotating 60 and translating 4.5 Å per step. Three steps, thus a rotation by 180 and a translational movement of 13.5 Å, are required to transfer three charges per subunit to open the channel (38). This relatively large scale movement of S4 was almost generally accepted as charge transfer mechanism in voltage-gated channels, until it was challenged by the helical-twist model, involving a much smaller movement, as shown in Fig. 3A a 180 rotation with no translational movement (39). Narrow, water-filled crevices make it possible to transfer the charges during gating. Fig. 3. Models of voltage-sensor movement. (A) Conventional model of voltage-sensor movement. Two opposing subunits are shown with the ion permeation path between them. Depolarization moves the extracellular portion of the S4 segment (red) outward through a short gating pore, opening the permeation pathway. Most of the S4 segment is surrounded by hydrophilic crevices/vestibules. The transmembrane-electric field falls mainly across the short gating pore. (B) Paddle model (Two opposing subunits are shown). Depolarization moves the paddle, the S3b helix and extracellular end of the S4 segment, outward through lipid, pulling the cytoplasmic activation gate open. The crystallization of the KvAP channel structure in which S4 was tightly bound to the C-terminal end of S3 (called S3b), and was located at the perimeter of the channel suggested another type of activation mechanism: the paddle mechanism (40). According to this model, the voltage sensor paddles are positioned inside the membrane, near the intracellular surface, when the channel is closed, and the paddles move a large distance across the membrane from inside to
7 Ion Channels Voltage-Sensing Mechanism 199 outside when the channel opens. This novel model immediately met strong criticism (41) due to its inconsistency with complementary experimental findings. For example, the paddle model cannot be generalized to all voltage-gated K channels, since in some channel types there are as many negative charges in S3b as there are positive charges in S4, making the S3b S4 paddle electroneutral (42). This electroneutrality would make the paddle unusable as a voltage sensor. Furthermore, the model assumes S4 to be at a considerable distance from the pore domain in open state, which stands in sharp contrast to the conclusions from electrostatic experiments (43) and to the conclusions from experiments showing that a cysteine at the N terminus of S4 can make a disulfide bond with a cysteine at the C terminus of S5 (44, 45). CONCLUSIONS Recent crystallization of several structures of voltage-gated channels allowed significant progress to be made towards understanding the voltage-sensing and gating mechanism. The most striking feature of these X-ray structures is the modular nature of the voltage sensor domain and its lack of extensive interactions with the pore domain (about 66% of molecular surface of the transmembrane region of each voltage sensor S1 S4 is exposed to lipids). Once the concept of relatively independent voltage sensing and pore domains is established, it brings the question of how and where they are coupled. For example, does the modular unit formed by a single S1 S4 anticlockwise helical bundle have the ability to function as a voltage-sensing electromechanical device on its own? What molecular interactions are responsible for the transduction of the voltage-sensing signal to another protein domain? What aspect of those interactions might be conserved across different systems? Hopefully future experimental and theoretical studies will bring an answer to these questions. REFERENCES 1. Hodgkin A, The conduction of the nervous impulse, Liverpool University Press, 1964, pp. 10 108. 2. Hille B, The superfamily of voltage-gated channels, in: Ion channels of excitable membranes (3 rd ed.), Sinauer Associates Inc., 2001, pp. 61 95. 3. Yu FH, Catterall WA, The VGL-chanome: a protein superfamily specialized for electrical signaling and ionic homeostasis, Sci STKE, 253, re15 (2004). 4. Ashcroft FM, Ion channels and disease (1 st ed.), Academic press, 2000, pp. 67 405. 5. Panyi G, Possani LD, Rodríguez de la Vega RC, Gáspár R, Varga Z, K + channel blockers: novel tools to inhibit T cell activation leading to specific immunosuppression, Curr. Pharm. Des., 18, 2199 220 (2006).
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