Control of Neuronal Excitability

Chapter 21

Uwe Heinemann

Istvan Mody

Yoel Yaari

Introduction

The behavior of nerve cells is determined by ionic channels. These are transmembrane proteins with a pore that permits ions to pass across the lipophilic membrane. Ion channels may be permanently open or regulated by stretch, voltage, or chemical compounds. Currents flow through these channels in an inward or outward direction dependent on the driving force, which is set by the difference between the actual membrane potential and the equilibrium potential for the given ion(s). An inward current depolarizes the membrane and is carried either by movement of positive charges (Na⁺, Ca²⁺) into the cell or of negative charges (Cl^-) out of the cell. Conversely, outward currents drive the membrane potential in a hyperpolarizing direction and are carried either by cation (K⁺) currents out of or anion (Cl^-) currents into the cell. At resting membrane potential, which is negative in all cells, inward and outward currents balance each other. The proteins that encode the water-filled, voltage-dependent ion channels are called α subunits. They are often associated with β, γ, and other auxiliary subunits. These may modulate properties of the channels, but they also regulate membrane anchoring and trafficking of ion channels. The receptor-gated ion channels usually consist of different α, β, and so on subunits, which all are required to form the ion channel. The nomenclature for ion channels is rather diverse. Physiologists and biophysicists originally named most ion channels according to their ion selectivity and dependence on voltage, ligand binding, or role in cellular function such as stretch, generation of receptor potentials, and so on. A second nomenclature often derives from studies of mutations in the fruit fly Drosophila. Scientists who originally cloned ion channels developed yet another nomenclature, and, finally, human geneticists use yet another. This makes reading the original literature difficult and sometimes time-consuming. I find the International Union of Basic and Clinical Pharmacology (IUPHAR) compendia on ion channels rather useful in this respect because it gives all the names used in the literature for all voltage-gated ion channels. I will follow the 2005 nomenclature of ion channels proposed by the International Union of Pharmacological Sciences. The most comprehensive survey of properties of ionic currents and their physiologic meaning is given by Hille.⁶⁵

Receptor-gated ionic channels mediate the information traffic between cells and set the membrane potential as a function of changes in the intracellular milieu such as those of adenosine triphosphate (ATP), pH, or calcium. Voltage-gated ionic channels determine how a neuron integrates synaptic information and propagates it to another neuron or to effector organs. Voltage-gated channels regulate the membrane potential, influence the integrating properties of the dendrites and the discharge mode of a cell, and are responsible for the generation and propagation of action potentials. At the presynaptic terminals, they influence Ca²⁺ loading of the terminals, which is a prerequisite for transmitter release. It is important to note that the distribution of ion channels over the surface of a neuron is not homogeneous. Neurons are polarized cells, which often express in their dendrites other channels than in the soma, the axon, and the presynaptic terminals. Regulation of sorting and direction into axonal and dendritic transport in neurons is much less understood than in epithelial and endothelial cells, which are also polarized. The anchoring of ion channels at distinct sites within and outside the synapse is another important issue because it determines the strength of synaptic coupling and the integration properties of neurons. This has become clear because mutations in stargazing and other anchoring proteins can contribute to epileptogenesis.¹⁰² Similarly, proteins involved in vesicle cycling and other presynaptic functions can contribute to epileptogenesis.⁵

For ionic currents to flow, an electrochemical gradient must be provided, which depends on transport processes across the neuronal membrane. Changes in membrane potentials can be brought about by electrogenic active or secondarily active transport, and such processes frequently influence the responsiveness of neurons. Therefore, in this chapter, I first consider the operation of such ion transport mechanisms. Subsequently, I treat the properties of channels that control the resting membrane potential. I then describe voltage-operated ionic channels that account for neuronal excitability and different discharge modes of neurons. The excitability of neurons is also controlled by low-affinity subsynaptic γ-aminobutyric acid (GABA), glycine, acetylcholine, and glutamate ionotropic receptors and by high-affinity ionotropic or metabotropic receptors, which are often located at extrasynaptic sites.

Properties of Ion Transporters and Generation of Membrane Potentials

A simple way for ions to cross the plasma membrane is by means of energy-driven pumps, which use the energy from ATP to overcome the barrier imposed by the plasma membrane. Ion pumps are proteins responsible for generating and maintaining the concentration gradients of Na⁺, K⁺, Ca²⁺, H⁺, and Cl^- ions across the plasma membrane; of H⁺ across vesicular membranes; and of Ca²⁺ across the mitochondrial membrane and the endoplasmic reticulum. They bind ions on one side of the membrane, physically transport it across the bilayer, and release it on the other side. Because energy is expended in this process (ATP hydrolysis), it is possible for such active transporters to move ions against a concentration gradient. The energy for this transport comes directly from ATP. It is therefore important that the respiratory chain is intact. The Na,K-ATPase is formed

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by α and β subunits. Four α subunits and four β subunits have been identified, and two α and β subunits are necessary for the transport process.⁵⁵^,¹⁴⁵ The ATP-dependent ion transporters affect the electrical behavior of a cell in two ways: They set up the electrochemical ionic gradients that underlie current flow when voltage- or ligand-gated channels are activated. They also affect membrane potential because the transporters are often electrogenic. As an example, Na,K-ATPase transports three Na⁺ ions out of a cell in exchange for only two K⁺ ions. Hence, the Na,K-ATPase imposes a hyperpolarizing drive on the membrane potential and thereby drives the membrane potential in a negative direction. In fact, the reversal potential for this transporter is quite negative. The membrane potential of neurons and glia would sit very much negative to -100 mV; if not, conductances for Na⁺, K⁺, and Cl^- would clamp the membrane potential to more depolarized potentials. In fact, it is the Na-K pump that causes K⁺ to accumulate within cells and sets up the gradient for Na⁺ to enter cells once Na⁺-permeable channels are open.¹³³ Because under resting conditions mostly K⁺ channels are open, K⁺ ions tend to leave the cells along their chemical gradient. This is prevented by the retaining force set up by negatively charged proteins. Thus, an equilibrium between influx and outflux of K⁺ is generated. This equilibrium is described by the Nernst equation. A membrane potential dominated by the K⁺ concentration gradient over the membrane would be close to -90 mV. This situation is approached in many astrocytes,¹¹⁶ which therefore respond to a change in extracellular K⁺ concentration more effectively than do neurons.

FIGURE 1. A: Active transporter. Principles of action of the electrogenic Na⁺,K⁺-ATPase. One adenosine triphosphate (ATP) molecule is used to transport three Na⁺ ions against the concentration gradient out of the cell, and two K⁺ ions are transported against the concentration gradient into the cell. B: Secondarily active transporter. In the example of this antiporter, three Na⁺ ions are moved along the electrochemical gradient into the cell, and one Ca²⁺ ion is moved against the electrochemical gradient out of the cells. Note that all transporters have a reversal potential. ADP, adenosine diphosphate.

Most neurons and muscle cells have membrane potentials more positive than -90 mV. This is because channels permeable to Na⁺ and Cl^- are open under resting conditions. These conductances are much smaller than those for K⁺. Nevertheless, the existence of Na⁺ leak channels implies that for each K⁺ ion that leaves a nerve cell, one Na⁺ ion can enter. The cells would slowly depolarize to zero if the Na-K pump did not become activated on intracellular accumulation of Na⁺ and thus restore the concentration gradients. Thus, the Na-K pump ultimately is responsible for the generation of the Na⁺ and K⁺ concentration gradients and the membrane potential. The Na-K pump can be activated by accumulation of both extracellular K⁺ and intracellular Na⁺.⁵⁸ During a seizure resulting from activation of Na⁺ and K⁺ channels, Na⁺ accumulates within the neurons and K⁺ in the extracellular space. This leads to activation of the electrogenic Na⁺ pump and then to a hyperpolarizing drive. This hyperpolarizing drive contributes to the termination of seizures and is responsible for the long-lasting afterhyperpolarization that follows a single seizure.⁶¹ When Na⁺ pumps lose their efficacy, afterhyperpolarizations become smaller, and such loss of efficacy may well underlie episodes of status epilepticus. Indeed, it has been shown that accumulation of intracellular Ca²⁺ can impair the function of the pump.⁵⁰ This may be caused by depolarization of mitochondrial membranes, which interferes with the generation of ATP,⁸⁹ by consumption of ATP in pumping Ca²⁺ out of cells and into intracellular stores, or by a direct modulation of Na,K-ATPase.

The electrochemical gradient set up by such pumps can be exploited by secondarily active transporters. These use the electrochemical force for inward or outward movement set up by active pumps to transport a second molecule against the concentration gradient. Such transporters import glucose, amino acids, and other agents into the cells as well as export metabolites, calcium, and protons. Often these transporters are not electrically neutral, and thus they can influence the membrane potential (FIGURE 1).

Secondarily active pumps are also involved in regulating the intracellular and extracellular ionic environment. They can use the electrochemical gradient for one ion species to move another ion in the same direction (cotransport) or in the opposite direction over the membrane (antiport). Such transporters do not affect the membrane potential if they are electrically neutral. However, in many cases the transporter is not electrically neutral. For example, the Na⁺-Ca²⁺ exchanger in most cases transports three Na⁺ ions into the cell against one Ca²⁺ ion out of the cell, thus providing a depolarizing drive to the cell.¹³^,¹⁴

Secondarily active transporters have a reversal potential. Thus, when the membrane potential moves beyond the reversal potential, the transport direction is also reversed. For example, the reversal potential for the Na⁺-Ca²⁺ exchanger is somewhere around -30 mV. This implies that depolarizations beyond this potential would drive Ca²⁺ into the cell, and there is indirect evidence that this occurs during spreading depression and anoxic depolarization.⁸⁴ Because the transmembrane ionic gradients strongly change during such conditions, however, the reversal potential also shifts. It is therefore necessary to consider in any of these conditions the actual reversal potential for a given transporter. This also applies to situations in which GABA or glutamate is released by reversed transport from nonvesicular compartments.²^,³

The intracellular Cl concentration is also affected by secondarily active transporters and in addition by Cl channels. Early during neuronal development, the NKCC1 transporter sets the Cl equilibrium potential above the resting membrane potential of neurons.¹¹⁹ As a result, inhibitory transmitters such as GABA and glycine produce depolarizations that early during development are sufficient to excite neurons.²⁸ These GABA-driven giant depolarizing potentials are part of early spontaneous depolarization waves in most neuronal structures.¹ Later in life (in humans, likely during the last trimester of embryonic development⁸³), the NKCC1 transporter is replaced by the KCC2 transporter, which extrudes Cl from cells and sets the reversal potential of Cl to values below the resting membrane potential, probably dependent on certain trophic factors.⁸² Even then, however, the direction of transport is regulated by the extracellular potassium concentration, and it may reverse

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transport direction at about 5.5 mM, resulting in a depolarizing Cl equilibrium potential.⁷⁶

Ion Channels Determining Resting Membrane Potentials

The K channels that most strongly contribute to generation of resting membrane potentials are likely two-pore domain potassium channels consisting of four transmembrane loops with two pores.⁵⁷ The K_2P channels are encoded by KCNK genes, with 11 members so far of the family expressed in the brain. These channels can be influenced by stretch, protons, oxygen tension, and lipids (e.g., arachidonic acid, anadamides), but also by volatile anesthetics and drugs such as riluzole, quinine, quinidine, and bupivacaine, as well as barium at concentrations of 1 to 2 mM.⁹² K_2P channels are outwardly rectifying and are likely a major route by which potassium is released from cells when they depolarize.

The resting membrane potential is also influenced by potassium inward rectifying (Kir) channels. Seven families of these channels are encoded by KCNJ genes.⁹⁰ These consist usually of four subunits with two transmembrane segments. These channels rectify because Mg and polyamines block the outflow of potassium through them. Kir channels frequently increase conductance when K accumulates outside the cells. Strongly rectifying Kir 3 family members are activated by G proteins through βγ subunits and weakly rectifying Kir 6 channels by the ratio of ATP to adenosine diphosphate (ADP). Kir 2 channels are expressed apart from neurons on muscle cells and are involved in vasodilation when K accumulates outside smooth muscle cells.²⁹ Kir 4.1 channels (perhaps together with Kir 5.1) form the astrocytic K channels involved in spatial K buffering.⁷⁴ These channels are very sensitive to Ba in low concentrations. This is in contrast to Kir 7 family members, which are only blocked by Ba at high concentration of about 100 μM. Studies on weaver mice suffering from epilepsy⁷⁵ have clarified that mutations in the selectivity filter in the pore-forming loop lead to permeability also for sodium ions and resulting depolarization when Kir 3.3 channels are activated by G proteins.

The ion channels that confer Na and Ca permeability to the membrane at resting membrane potential are not well understood. Some transient receptor potential (TRP) channels may be involved.³⁰^,¹¹⁵ These are rather unselective cation channels with little rectification, which are thought to be involved in receptor potential generation. However, some of them are widely distributed throughout the brain. Thus, all TRPC member channels are expressed in the brain. They are usually activated by the Gq type of G proteins, diacylglycerol (DAG), and also neurotrophic factors. TRPV1–4 channels are expressed not only in peripheral, but also in many central neurons. TRPM2–4 channels are also expressed in brain and are modulated by arachidonic acid, redox state, and cytokines.

Ion channels that likely also influence membrane resting potential are the cyclic nucleotide–regulated ion channels.⁶⁹ They are rather unselective cation channels permeable for both sodium and potassium. They play an important role in phototransduction, but many of them are also expressed in central neurons. They are regulated by intracellular nucleotides such as guanosine 3′,5′-cyclic monophosphate (cGMP) or cyclic adenosine 3′,5′-monophosphate (cAMP) and show little voltage dependence. This is in contrast to the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which are also relatively unselective cation channels. These ion channels are activated by hyperpolarization and shift the membrane potential back toward resting membrane potential. The activation curves for these ion channels are regulated by cAMP and cGMP. There are four subunits of these ion channels, all of which are expressed in the brain. They are frequently involved in membrane potential oscillations and are also important for rhythmogenesis in thalamic neurons and other neurons displaying membrane potential oscillations. Cells that express these channels often show resonance properties: This implies that neurons become especially sensitive to specific frequencies of synaptic input usually in the θ and α frequency ranges. Upregulation of such channels may be involved in homeostatic plasticity but also following hyperthermia-induced convulsions.²⁶

Which chloride channels are contributing to resting membrane potential is unclear. Two calcium-dependent chloride channels have been cloned from rat brain, but their functional role is unclear. Whether-volume activated chloride channels are expressed in neurons is unclear, but such channels could be involved in osmoregulation of astrocytes.

Voltage-Gated Ionic Channels

These channels determine the excitability of neurons, the different firing modes, the integrating properties of dendrites, and the mechanisms that lead to transmitter release from presynaptic terminals. Except for voltage-dependent chloride channels, most voltage-gated ion channels belong to one superfamily.¹⁶⁰ They all build on a pore-forming unit contained in the α subunits. Voltage-regulated Na and Ca channels consist of four homologous motifs with six transmembrane α helices termed S1 to S6 with a membrane reentrant loop between S5 and S6. The voltage-gated potassium channels consist of four α subunits, which combine to form a voltage-regulated potassium channel. As in voltage-gated Na and Ca channels, the ion-conducting pore and selectivity filter are formed by the S5 and S6 segments and the reentrant pore loop between them. Four subunits are also used to form cyclic nucleotide–gated (CNG), HCN, and TRP channels. Heteromeric assembly of different subunits, combinations with different auxiliary subunits, RNA editing, and splicing confer very different properties to these channels.

FIGURE 2. Reaction schemes of voltage-gated channels. A: Principle of action of transient Na⁺ or K⁺ channels. At rest, the channel is closed by the position of a gate in the outer mouth of the channel (closed, activatable). On depolarization, the channel proteins change their conformation, permitting passage of ions. The conformation change is controlled by a voltage sensor that measures the potential difference across the neuronal membrane. On further depolarization, an inner, ball-like structure moves into the inner mouth of the channel, obstructing the passage of ions (closed, inactivated). Hyperpolarization is required to change the conformation from the inactivated state to the activatable state. B0, B1, B2: Summary of typical reaction schemes of voltage-operated channels. C, closed; CA, closed activatable; CI, closed inactivatable; D, depolarization; H, hyperpolarization; O, open.

Voltage-gated ionic channels are thus membrane-spanning proteins that form a pore; the opening and often also the closing of the pore are regulated by the transmembrane voltage gradient. The voltage sensor is contained in the S1-to-S4 segment of the α subunits, forming the channel, which is absent in Kir and K_2P channels. Voltage-gated ionic channels usually have auxiliary subunits.¹⁶⁰ The voltage-gated sodium channels possess only four β subunits, which modulate channel activation and regulate membrane surface expression. Therefore not only mutations in α subunits but also those in β subunits can lead to epilepsy. Voltage-gated Ca channels are regulated by β, γ, and α2δ subunits. The four β subunits are all intracellular proteins that regulate channel gating and surface expression. Mutations in β subunits can thus also lead to channelopathies, including epilepsy. There are eight γ subunits including stargazing,¹⁰² which link the Ca channels to other transmembrane proteins, including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors. The α2δ subunits are binding sites for anticonvulsants such as pregabalin and gabapentin.³⁹

Voltage-regulated K channels have different types of auxiliary subunits.⁹³ K_V1 channels are associated with β1–3 subunits; K_V4 channels with Kchip 1–4; and K_V3, 4, 7, 10, and 11 channels with the five members of mink-like subunits.¹⁶⁰ The β subunits in the K_V1 channel family influence channel closing. Kchips belong to the superfamily of Ca sensor proteins and regulate channel kinetics and channel membrane surface expression. The mink-like subunits are also important regulators

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of channel dynamics. Finally, the K_V2 family is modulated by K_V5, 6, 8, and 9 modifier/silencer subunits.

The gating of these ionic channels occurs preferentially at a defined membrane potential at which the probability of channel opening increases strongly. However, because the sensor for the gating is located within the membrane, it recognizes the voltage difference between the inside and outside leaflets of the membrane. On both sides of the membrane, there are negative surface charges that attract preferentially Ca, Mg, and H ions. Consequently, changes in H⁺, Ca²⁺, and Mg²⁺ and proton concentration can influence the gating properties.⁶⁶ Occasionally, these ions can also modify permeability properties. Well-known examples are cation channels, which can become activated during rapid decreases in Ca²⁺ concentration.⁶³^,¹⁵⁶

In addition, ionic channels can be influenced by intracellular metabolism. For example, certain K channels are rather sensitive to the ATP content of a cell. At physiologic ATP levels, these channels are closed, whereas depletion of ATP leads to opening of K channels and consequently to hyperpolarization. Block of such channels can induce seizures.⁷ Some ionic channels possess redox sites such as voltage-gated K channels and K_2P channels, which react to the formation of free oxygen radicals or are sensitive to arachidonic acid and its derivatives.¹⁰³ Most ionic channels change their properties on phosphorylation, and many protein kinases and some phosphatases can therefore affect the opening and closing of voltage-gated channels. In some cases, such modulations give the cells distinctly different discharge modes, whereas in others, there is a continuum of modifications in the reactivity of a given neuron. In addition, the intracellular level of Ca²⁺ can cause activation and occasionally inactivation of ionic channels.

Based on permeability of the pore, the resulting transmembrane currents are differentiated as Na⁺, K⁺, Ca²⁺, and Cl^- currents. Proton currents have also been described and are a prerequisite for formation of radical oxygen species released from activated microglial cells.⁴³ Within these different species of ionic currents, a distinction is made between persistent and transient currents. Most of the persistent channels open on depolarization and some on hyperpolarization of the membrane potential with respect to the resting membrane potential. Such channels behave according to the reaction schemes illustrated in FIGURE 2B. When persistent ionic currents are activated, the channels open and close, with an increased probability of being open for the whole period of time that the membrane potential is above the threshold for channel opening.

Transient currents show a time-dependent variation of probability of being open when the membrane potential is above that required for channel gating. Their reaction scheme can be formally described as in FIGURE 2A. In these channels, the open probability strongly decreases with time. This time-dependent inactivation is often mediated by a ball formed by the intracellular end of transmembrane proteins. These balls move into the inner mouth of the channels, thereby preventing further passage of ions. This type of inactivation is designated chain-and-ball inactivation and resides at the N terminal of the channel protein.⁶⁶ By contrast, there is also a C-type inactivation, which depends on conformational changes in the channel itself. Inactivation of either type makes the channel temporarily refractory and induces a time-dependent short-term memory into channel properties. The conditions and time it takes to remove inactivation are usually studied in paired pulse experiments. Removal of inactivation in sodium channels is typically delayed when anticonvulsants such as carbamazepine or phenytoin are applied.¹¹⁸ Besides the kinetics of an ion channel, the steady-state behavior also is used to describe the channel properties.⁶⁸ Steady-state inactivation curves are determined by current measurements with pulses evoked from different membrane potentials to a potential at which the current is maximal. Conductances are then derived from Ohm’s law by taking into account the driving force for the ion species and the measured current amplitude. Normalized conductance curves are then plotted and give the percentage of ion channels that can be maximally opened as a function of resting membrane potential. The steady-state activation curve describes the relative conductance as a function of the membrane potential achieved during a voltage step. A leftward shift of the steady-state

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inactivation curve then indicates that at a given membrane potential a fewer channels are available for current generation. Such leftward shifts of steady-state inactivation curves contribute to effects of anesthetics and anticonvulsants. A leftward shift of the steady-state activation curve indicates that more channels can contribute to an ionic current and for the sodium channel would indicate a lower threshold for the generation of action potentials.

Thus, voltage-gated ionic channels can frequently be modulated by drugs. These often affect membrane fluidity, changing the energy barriers that a channel protein must overcome to change its conformation from a closed to an open state. Moreover, drugs can bind to channel proteins, stabilizing a given conformation and changing its threshold for activation. Especially interesting are drugs that are use dependent, that is, they bind to a channel protein only when it is activated. Such drugs often enter the open mouth of an ionic channel and then move very slowly through the channel, preventing further current flow. In some cases, they affect the transition from an inactivated to an activatable state and thereby also produce a use-dependent effect. On the other hand, some drugs and toxins impede the inactivation process, changing a transient into a persistent current.

Voltage-Gated Sodium Currents

The fast transient Na⁺ currents are mainly involved in the generation of action potentials, first described by Hodgkin and Huxley.⁶⁷ The rapid upstroke of the action potential is the result of a voltage-dependent increase in membrane Na⁺ conductance. The threshold for action potential generation is defined as the membrane potential at which Na (and Ca) conductances equal those of K (and Cl) conductances. This implies that Na channels must also be active in the subthreshold range. In most central neurons, the threshold of activation for the fast Na⁺ current is about -60 mV.²³ The current–voltage relationship of this current is characteristically a U-shaped curve, for which the maximum of current amplitude is usually reached at about -20 mV. The fast transient Na⁺ currents inactivate rapidly. The inactivation behavior of the current has been described with time constants of 2 to 4 msec in most neurons studied. The Na channel can only reactivate when inactivation is removed. This is a voltage-dependent and time-dependent process often involving two time constants.¹¹⁸ Rapid removal of inactivation is typical for interneurons, which therefore can fire in higher frequencies than most glutamatergic neurons. By means of electrophysiologic and biochemical techniques, several toxins have been identified as blocking Na⁺ channels. After it was found that tetrodotoxin (TTX) acts in nanomolar concentrations as a selective blocker of most neuronal Na⁺ channels, it became a useful chemical tool for studying properties of these channels in more detail.⁸⁰ In addition to TTX-sensitive channels, TTX-resistant Na⁺ channels have been detected.¹⁵⁹ Acutely isolated neurons from the medial entorhinal cortex of the rat exhibit a TTX-resistant Na⁺ current in addition to the TTX-sensitive Na⁺ current.¹⁵⁴ Like TTX, saxitoxin and μ-conotoxin are able to block Na⁺ conductance by either occluding the channels or causing conformational changes.³¹ Moreover, a number of lipid-soluble alkaloid toxins, including batrachotoxin, veratridine, aconitine, and grayanotoxin, can shift the voltage dependence of activation in a hyperpolarizing direction and prevent current inactivation, resulting in persistent channel activation at normal membrane potentials.²⁴ The voltage dependence of Na⁺ currents is also changed by the application of β-scorpion toxins, which cause enhanced activation. Brevetoxins and ciguatoxins can cause repetitive firing of nerve cells by shifting the activation curve of Na⁺ currents to more negative potentials.²⁴ Toxins such as veratridine and brachatoxin can therefore readily induce convulsant activity.¹⁰⁷

Sodium currents are encoded by nine different α subunits, which combine with four different β subunits. Na_V1.1–1.3, 1.5, and 1.6 are expressed in central neurons. Na_V1.1 (SCN1A) channels associate with β1 to β4 subunits.²⁴ Na_V1.1 channels are highly expressed in hippocampal interneurons, and deletion of this channel leads to reduced firing of interneurons in the hippocampus, which explains some of the hereditary epilepsies associated with mutations in these channels such as generalized epilepsy with febrile seizures plus (GEFS+) and myoclonic epilepsy. The β4 subunit in this and other channels accelerates removal of inactivation, permitting high-frequency generation of action potentials, as is typical for many interneurons. Na_V1.2 (SCN2A) can also interact with all β subunits. It is highly expressed in axons of central neurons. Mutations in these channels have also been linked to epilepsy.¹⁴³ Na_V1.3 channels are highly expressed during development but are also expressed on somata in adult brain cells. These channels are upregulated after nerve injury and, due to rapid removal of inactivation, can be involved in high-frequency axonal discharges. This implies that the action potential frequency in somatic recordings may not necessarily be the same in axon terminals. They interact with β1 and β3 subunits. Na_V1.5 channels (SCN5A) are insensitive to TTX and, besides heart muscle cells, are also expressed on subgroups of central neurons. They can interact with all four β subunits. Finally, Na_V1.6 (SCN8A) channels are expressed in central neurons and mostly on the somatodendritic region of output cells in the cerebral cortex, hippocampus, and cerebellum. It is thought that these ion channels contribute at least partially to persistent sodium channels.

The β subunit 1 occurs with different mutations, leading to GEFS+ and also to temporal lobe epilepsy (TLE).⁷³ Persistent noninactivating Na⁺ current has been found in a number of neurons.⁴^,⁶^,⁴⁸ Its activation threshold was determined at potentials more negative than that of the fast transient Na⁺ current, at about -70 mV. Because of activation near the resting membrane potential of neurons and the loss of inactivation, this current can contribute to subthreshold membrane potential oscillations and resonance, playing an important role in the genesis of the θ rhythm in these cells.⁴ It is likely that also some metabolites can remove inactivation and thereby permit generation of persistent sodium currents. Another possibility is that persistent sodium channels are due to window currents. When the steady-state activation and inactivation curves are considered, they often overlap and generate a voltage range in which a portion of these channels is frequently open.

Voltage-Gated Calcium Currents

Calcium currents play a major role in neuronal excitability, directly by their contribution to membrane depolarization and indirectly through the elevation of the intracellular concentration of free Ca²⁺. Voltage-dependent Ca²⁺ conductances in neurons contribute to the generation of dendritic spikes, slow somatic depolarizations, and related burst discharges.²² Influx of Ca²⁺ through voltage-gated Ca²⁺ channels can activate Ca²⁺-dependent K⁺ channels and regulate many intracellular Ca²⁺-dependent processes. Calcium channels are also responsible for rapid delivery of Ca²⁺ to trigger transmitter release.⁴⁶^,¹⁵⁰

Voltage-gated Ca²⁺ channels consist of α1 subunits with which β subunits, α2δ, and γ subunits can be associated.²⁵ In the classic studies of sensory neurons by Carbone and Lux,¹⁹ they showed that voltage-gated Ca²⁺ channels could be differentiated into two major categories: (a) low-voltage-activated (LVA) and (b) high-voltage-activated (HVA)

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channel types. The α1 subunits of Ca channels can be differentiated into three families: (a) Ca_V1, (b) Ca_V2, and (c) Ca_V3 members.²⁵ The Ca_V1 family shares high threshold of activation, no voltage-dependent inactivation, and sensitivity to dihydropyridines, and these are often referred to as L-type Ca channels. In the central nervous system (CNS), only Ca V 1.3 (α1c, CACNA1C) channels are expressed on somata and dendrites of neurons, with little effect on regulation of transmitter release. The Ca_V2 subfamily (Ca_V2.1–2.3) has three members, which are all insensitive to dihydropyridines. Ca_V2.1 (α1A, CACNA1A, or P/Q-type) channels are involved in transmitter release but are also found in many neurons expressed on dendrites. They are sensitive to ω-agatoxin IVa. The Ca_V2.2 (α1B, CACNA1B, or N type) channels are also expressed presynaptically and on dendrites. These channels show time-dependent inactivation and are blocked by ω-conotoxin GVIA. Like Ca_V1 channel members, these two channels require high levels of depolarization to become activated. Ca_V 2.3 channels (α1E, CACNA1E, or R-type channels) require less strong depolarization for activation, are expressed on somata and dendrites, and are sensitive to the peptide SNX-482. They can be involved in epilepsy.¹⁴⁴ Low-voltage-activated channels contain Ca_V3.1 to 3.3 members (α1G, H, I; CACNA1G, H, I), are rather sensitive to low concentrations of Ni, and form the T-type Ca channels. In most neurons, these channels activate at potentials positive to -70 mV. Inactivation of the T-type Ca²⁺ channels develops monoexponentially within tens of milliseconds and shows a strict dependence on voltage. Unlike in L-type Ca channels, the inactivation kinetics is independent of Ca²⁺ influx through the channel.²⁰^,²¹ No specific high-affinity antagonist has so far been identified for the T-type Ca²⁺ current, although antiepileptic drugs used to treat petit mal, such as ethosuximide and dimethadione, reduce T-type Ca²⁺ currents in thalamic neurons and dorsal root ganglion cells.³⁵

The behavior of voltage-gated Ca currents is influenced by different subunits. The intracellular β subunit consists of four known isoforms β1 to β4 (CACNB1–CACNB4). Mutation in β4 subunits also are involved in ataxia and epilepsy in mice and humans.¹⁸ The β subunits regulate current density by controlling the amount of α1 subunit expressed at the cell membrane. In addition to this trafficking role, the β subunits regulate the activation and inactivation kinetics and shift the voltage dependence for activation of the α1 subunit pore in the hyperpolarizing direction.

The α2δ subunits are formed from a distinct gene. The α2 subunit is the extracellular glycosylated subunit that interacts the most with the α1 subunit. The δ subunit has a single transmembrane region with a short intracellular portion, which serves to anchor the protein in the plasma membrane. There are four α2δ genes: CACNA2D1 to CACNA2D4. Coexpression of the α2δ enhances the level of expression of the α1 subunit and causes an increase in current amplitude, faster activation and inactivation kinetics, and a hyperpolarizing shift in the voltage dependence of inactivation.¹⁴⁶ Some of these effects are observed in the absence of the β subunit, whereas in other cases the coexpression of β is required. The α2δ-1 and α2δ-2 subunits are the binding sites for at least two anticonvulsant drugs, gabapentin and pregabalin, which also find use in treating chronic neuropathic pain.³⁹

The γ subunits are associated with only some of the α1/β complexes. The γ subunits do not affect trafficking and for the most part are not required to regulate the channel complex. There are eight genes for the γ subunit: CACNG1 to CACNG8. The γ1 subunits are expressed in skeletal muscle, and the γ2 and γ3 subunits may be associated with the P/Q- and N-type channels. Some γ subunits (γ3, γ4, and γ8) and particularly stargazing (γ2) associate Ca channels with AMPA-type glutamate receptors.¹⁴⁸^,¹⁴⁹

Voltage-Gated Potassium Currents

Whereas voltage-gated sodium and calcium channels are formed by a distinct subgroup of proteins forming the α subunit, most voltage-dependent potassium channels are formed by four subunits.⁶⁰^,¹⁶⁰ The first cloned potassium channels were detected in Drosophila and named shaker (K_V1.1–1.8; KCNA1–7, KCNA10), followed by shab (K_V2.1 and 2.2; KCNB1.2), shaw (K_V3.1–3.4; KCNC1–4), and shal (K_V4.1–4.3; KCND1–3) related subunits. The M-type potassium channels modulated by muscarine were originally detected in sympathetic ganglia and form the K_V7 family with five members (KCNQ1–5). The identification of the voltage-dependent potassium channels forming the K_V10 to 12 families (eag, erg, and elk; KCNH1–8) was based on the identification of the ether-a-go-go channel in Drosophila. In the different families, the same subunits can form homomeric potassium channels, or different subunits can combine to form heteromeric channels. Heteromeric channel formation is well documented for K_V1, K_V7, and K_V10 families but likely also occurs in other families of voltage-gated potassium channels. The combination of subunits in heteromeric channels frequently confers different properties to the ion channels than those expected from the characteristics of homomeric potassium channels in a given family. The diversity of potassium channels is further increased by silencer and modifier subunits belonging to the K_V5, 6, 8, and 9 families. These subunits do not form conducting channels but can combine with members of the K_V2 family to form functional channels with different properties than those observed in homomeric K channels from this family. As with Na and Ca channels, β subunits have been identified that can combine with members of the K_V1 and K_V2 families to provide for altered channel properties but also likely are involved in channel trafficking and surface expression. Kchip1 can associate with K_V4 family members and modify their properties. Calmodulin interacts with K_V10 members and minK proteins with Kv11 family members. In addition, alternative splicing confers different properties to K channels.

Thus a wide variety of K⁺ currents exists in excitable cells, and potassium channels represent the most diverse type of voltage-gated ion channels known, comparable only to GABA_A receptors. Apart from their genetic identity, potassium currents can be distinguished by their voltage sensitivity, kinetics of activation and inactivation, single-channel behavior, and pharmacologic modulation. With respect to these properties, macroscopic voltage-gated K⁺ currents found in neurons can be subdivided into two types: (a) outward delayed rectifying K⁺ currents and (b) (fast) transient K⁺ currents.

FIGURE 3. Outward K⁺ currents activated by depolarizing voltage changes preceded by a hyperpolarizing prepulse. Currents were recorded in an acutely isolated entorhinal cortex cell. A: Currents were evoked by application of depolarizing voltage commands from the holding potential of -60 mV to potentials between -60 and -30 mV for 200 msec following a hyperpolarizing prepulse to -110 mV for 1,000 msec. B: Steady-state activation and inactivation curves for isolated fast transient A-type currents (closed lines) and delayed rectifying currents (dotted lines). Overlap of steady-state inactivation and activation curves indicates the voltage range in which channels open and close permanently. Note that around resting membrane potentials A-type currents can contribute to the stabilization of membrane potential.

Most of the delayed rectifying K⁺ currents (I_K) have a relatively high activation threshold. The currents activate at potentials more positive than -40 mV.⁴⁷^,⁶⁸^,¹³² Therefore, I_K seems to activate only during action potentials, not in the subthreshold voltage range. Hence, I_K contributes to spike repolarization.¹³⁹ In addition, I_K may be partly responsible for spike broadening during repetitive firing, due to slow accumulating current inactivation. In many neuronal preparations, time constants of 2 to 5 seconds have been determined to describe the inactivation behavior of this current (FIGURE 3).⁸^,¹⁰⁵ Pharmacologically, these currents are sensitive to tetraethylammonium (TEA). Delayed rectifier currents can be encoded by most members of the K_V1 family, which are all expressed in the central nervous system except for K_V1.7. Except for K_V1.4, they all encode for delayed rectifier-like ion channels. Members of the K_V1.1 and 1.2 families share a relatively high sensitivity to kaliotoxin and to dendrotoxin. In addition, K_V1.6 is sensitive to dendrodotoxin. Delayed rectifier channels can also be encoded by K_V2 family members, which are modulated by a number of accessory

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subunits. Their pharmacology is relatively sparse. They are sensitive to TEA and show only low sensitivity to 4-aminopyridine (4-AP). It is interesting that K_V2.2 is sensitive to phencyclidines, which are generally known to cause open channel block in N-methyl-D-aspartic acid (NMDA) receptors, and otherwise are used for studies on NMDA receptors.⁴⁹ Finally, members of the K_V3 and K_V4 families can encode delayed rectifier currents. K_V3 family members encoding delayed rectifier currents are rather sensitive for 4-AP, whereas K_V4 family members show low sensitivity. The K_V3 family members are all expressed in central neurons except for K_V3.3. It is interesting that K_V3.1 and 3.2 encode for delayed rectifier currents in many interneurons. They are sensitive to relatively low levels of TEA and 4-AP. On one hand, these drugs prolong action potentials in interneurons, but on the other hand, they strongly reduce firing frequency and thereby GABA release. It is believed that these effects contribute to the convulsivogenic effects of TEA and 4-AP in low concentrations.

Fast transient A-type currents (I_A) markedly differ from I_K in their kinetics; they show a fast activation following depolarization and inactivate rapidly, within tens of milliseconds.⁴⁷^,⁸⁶^,¹⁰⁴ Their threshold of activation is at about -45 to -60 mV, that is, at a more hyperpolarized level of membrane potential than that of I_K⁷¹ and below the threshold for action potential generation. Moreover, at membrane potentials more positive than -50 to -30 mV, A-type currents cannot be activated because they show a strong steady-state inactivation behavior. In most neurons, half-maximal inactivation for A currents was determined at potentials between -85 and -70 mV.¹⁰⁶ The pharmacologic sensitivity of I_A is also different from that of I_K. The A-type current is selectively blocked by 4-AP in the micromolar (K_V1 and K_V3 family members) to millimolar concentration range (K_V4 family members), but it is relatively insensitive to TEA. In hippocampal neurons, the I_A can also be blocked by low concentrations of the snake toxin dendrotoxin.¹⁰⁰

It has been shown that I_A plays an important role in determining the onset of discharge in response to a depolarizing stimulus and in the regulation of repetitive firing.³²^,¹⁰⁴ In addition, I_A seems to be involved in repolarization of the action potential because the spike is broadened when I_A is blocked by 4-AP.¹⁴¹

In a few reports, a K⁺ conductance (I_D) has been characterized that activates rapidly within milliseconds, like I_A, but inactivates slowly over several seconds.⁵⁹^,¹⁴⁰ In contrast I_K, this current is insensitive to TEA, but it is very sensitive to 4-AP and dendrotoxin. These potassium conductances seem to be responsible for a longer delay in the onset of firing in response to long-lasting depolarizing stimuli. Because I_D activates rapidly on depolarization, it tends to keep the cell from depolarizing further. Only as I_D slowly inactivates does the cell reach threshold and fire after delay, which can have a duration of up to about 15 seconds.¹⁴¹ The rapid activation of I_D suggests that it may also participate in spike repolarization.

In the K_V1 family (KCNA1–7, KCNA10) only K_V1.4 (KCNA4) encodes for an A-type current. K_V1.4 is highly expressed on mossy fibers and axons, and it is believed that K_V1.4 alone or in combination with K_V1.2 controls presynaptic transmitter release in many central neurons. Indeed, 4-AP in relatively low concentrations increases presynaptic Ca uptake in Schaffer collaterals and mossy fiber terminals. For pharmacologic studies of effects of Ca channel blockers, it is noteworthy that many members of the K_V1 family are also moderately sensitive to L-type Ca channel blockers. One family member (K_V1.2) is also sensitive to picrotoxin. The A currents can also be encoded by K_V3 and K_V4 family members.

Whereas K_V3.1 and 3.2 genes (KCNC1, KCNC2) encode for delayed rectifier currents, that for 3.4 encodes for transient potassium currents with a relatively high sensitivity to TEA and for A-type potassium currents in hippocampal granule cells. The genes for the K_V4 channel family members (KCND1–3) all encode for A-type potassium currents with low sensitivity to 4-AP. They seem to be expressed in the somatodendritic region of pyramidal cells and regulate backpropagation of action potentials and modulate excitatory input to dendrites.⁷⁷ The expression of these channels is modulated by Kchip subunits, which also influence their kinetics.

The M-type current (I_M) is encoded by KCNQ genes and belongs to the K_V7 family. I_M is a small, subthreshold, voltage-dependent outward K⁺ current that is activated at more hyperpolarized potentials (positive to -60 mV) than the delayed rectifying current. The current activates and deactivates slowly and does not inactivate.¹⁵ Thus, as the only K⁺ current that both activates below the spike threshold and does not inactivate, I_M can play a unique role in the control of cell excitability. In addition, I_M contributes to the resting membrane potential in many neurons.³⁴^,³⁸^,⁹⁶ It also seems to contribute to the early spike-frequency adaptation and is involved in generation of the medium afterhyperpolarization, an up-to-100-msec undershoot that follows an action potential or a spike train.¹⁴¹ The M current can be completely suppressed by muscarinic agonists and by specific blockers such as XE-991 or linopiridine. It is encoded by family members of the K_V7 family, also named KCNQ channels. It is of interest that heteromultimers of K_V7.2

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and 7.3 are usually expressed in central neurons and their axons. In addition, K_V7.5 also is expressed in central neurons. The expression of these channels is particularly high during development, when the expression of K_V1, 2, and 3 members is still low. This implies that K_V7 deletions or mutations in mice are often lethal. Recently a transgenic mouse has been made in which the expression of KCNQ channels can be downregulated at desired time points. These animals all develop epilepsy.¹¹² This channel family has been gaining in interest because relatively specific agonists such as retigabine are available that are powerful anticonvulsant drugs.⁴⁰^,¹²² K_V7 channels are strongly involved in rhythmogenesis and in resonance phenomena.¹¹²

Based on the Drosophila ether-a-go-go channels, three families of voltage-gated potassium channel families have been detected, most of which are also expressed in the brain.¹⁶^,⁶⁰ These belong to the K_V10 (KCNH1 and KCNH5; EAG1 and EAG2), K_V11 (KCNH2, KCNH5, and KCNH7; erg1 to erg3), and K_V12 (KCNH8, KCNH3, and KCNH4; elk3, elk2, and elk1) families. Most of the family members are expressed in brain except for perhaps K_V12.3. Some of the family members are activated at very low membrane potentials and therefore can contribute to resting membrane potential. Their activation can be very slow. K_V10.1 is a potassium channel that is also permeable for calcium ions and is blocked by calcium/calmodulin.¹⁷ K_V10.2 is also blocked by intracellular calcium. It activates at –100 mV. The K_V10 family members are blocked by imipramine and by astemizole.⁵² The K_V11 members can form heteromeric channels. Because of the involvement of one of their members in the long-QT syndrome in heart disease, there is a wealth of pharmacologic agents available with which it should be possible to learn more about their functional role in central neurons. K_V12 channels activate at hyperpolarized membrane potentials with rather slow kinetics and are insensitive to 4-AP and TEA but sensitive to barium.

Calcium- and Sodium-Activated Potassium Channels

With respect to their kinetic properties and their sensitivity to various pharmacologic agents, at least three different types of Ca²⁺-activated K⁺ currents have been distinguished in neurons of the central nervous system. A Ca²⁺-activated K⁺ current of large single-channel conductance (150 to 300 pS) has been detected in most neurons studied.⁹¹ This current is also voltage dependent and contributes to spike repolarization and fast afterhyperpolarization. The channel is constituted by a K_Ca1.1 α subunit encoded by the KCNMA1 gene. These channels associate in the brain with one of three β subunits, β2 to β4. The β2- and β3-containing K_Ca1.1 channels are sensitive to charybotoxin and iberotoxin, and the β4-containing channels are not but are potentiated by 17β-estradiol. β2 and β3 subunits confer inactivation to these channels. It is of interest that a number of other activators exist for these channels that may have potentially anticonvulsant effects. These include drugs such as NS 1608, NS 1619, and BMS 204352.¹⁵³ Mutations in the β3 subunit have recently been associated with susceptibility to idiopathic epilepsies.

The second class of Ca-activated K currents belong to the K_Ca2 family (KCNN1–KCNN3), which has three members.¹⁵³ These SK channels are exclusively regulated by intracellular Ca and are not voltage dependent. Voltage-clamp studies have suggested that they encode medium-duration Ca²⁺-activated K⁺ currents with a single-channel conductance of 20 to 60 pS, which can contribute to the medium afterhyperpolarizations during spike trains and to afterhyperpolarizations that last for about 200 msec.¹¹¹^,¹³⁸ These channels are blocked by bicucullin methiodide and may contribute to the epileptogenic action of bicuculline. It is interesting that the SK-type channels can be activated by drugs such as EBIO, NS 309, and riluzole.¹¹⁰ The K_Ca3.1 channel (KCNN4) is not expressed on neurons but is expressed strongly on microglial cells and may contribute to neuronal damage during status epilepticus.¹²⁵ Blocking these channels prevents activation of inducible nitric oxide synthase (iNOS)- and nitric oxide (NO)-dependent neuronal damage.⁸¹ These channels are also activated by methylxanthines such as caffeine and theophylline.

A prominent Ca-dependent K current (I_AHP) accounts for the late afterhyperpolarizations that contribute to afterhyperpolarization following seizures. These currents are blocked by cAMP and consequently by norepinephrine through β receptors, dopamine through D1-like receptors, and through other agents that upregulate cAMP.⁶² These currents are resistant to TEA and also to other K_Ca channel-blocking toxins. Their molecular nature has not been identified.

Closely related to Ca-activated potassium channels are Na-dependent K channels named K_Ca 4.1 (SLACK, SLOW2.2, KCNT1) and K_Ca 4.2 (SLICK, SLOW2.1, KCNT2). These channels are already active at physiologic intracellular sodium concentration and Cl concentrations and can form heteromultimers in which the K_Ca4.2 subunit is dominant. These ion channels are regulated by G_αq-type receptors, which activate protein kinase C. This is of interest because mGluR1 and muscarinic M1 receptors use this pathway. The two subunits are differently regulated. Whereas K_Ca4.2 is suppressed by muscarinic agonists, K_Ca4.1-mediated currents are increased. Therefore these ion channels may contribute to the differential effects of muscarinic and mGluR receptor–mediated effects in different neurons. The sodium-dependent K conductances are only blocked by very high concentration of Ba and quinidine.¹⁵³

A further, not yet genetically identified ionic channel underlies cation currents activated by elevation in intracellular Ca concentration.¹⁰⁹ These so-called CAN channels are sensitive to flufenamic acid, which in hippocampal neurons was shown to affect depolarizing afterpotentials and paroxysmal depolarizations during seizure-like events.¹²⁴ The molecular identity of these ionic channels is not known, but it is likely that TRP channels are involved in these currents.

Finally, a cation current can be activated by lowering the extracellular Ca concentration, which may contribute to epileptogenesis because sometimes the extracellular Ca concentration drops to very low levels during seizure-like events.⁶³^,⁶⁴^,¹⁵⁷

Bursting Behavior

Spontaneous generation of seizures seems frequently to depend on the presence of burster cells. These are neurons that, when excited, produce an all-or-none response, consisting of a depolarizing envelope that triggers a burst of action potentials. Such burster neurons have been described in the neocortex,³³ hippocampal areas CA3 and CA1,¹⁵⁵ and the subiculum.¹¹ The area most abundantly equipped with burster neurons is the subiculum, where about 50% of pyramidal cells have bursting properties. In the neocortex, the hippocampus, and the subiculum, the bursting behavior is caused by a persisting Na⁺ current, whereas in CA1 pyramidal cells of epileptic animals, the bursting behavior seems to depend on T-type inward Ca²⁺ currents.¹⁵⁸ The notion that persistent Ca²⁺ currents are involved in ictogenesis is supported by the observation that Ca²⁺-channel blockers possess anticonvulsant properties. In the concentration ranges in which these agents have anticonvulsant effects, however, they may no longer be specific for Ca²⁺ channels.¹³⁵ On the other hand, many anticonvulsant drugs

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not only affect the kinetics of fast Na channels, but also block persistent Na channels and some Ca channels.¹²⁰

Membrane Potential Oscillations and Resonance Behavior

Voltage-gated currents are also involved in the synchronization of neurons, which is necessary for the generation of epilepsy. The finding that drugs that suppress primarily generalized nonconvulsive seizures, such as absences, block T-type Ca²⁺ currents has made this very clear.³⁴ This phenomenon is discussed in Chapter 31.

A second current apparently involved in synchronization is I_Q (I_H, I_F), which is encoded by HCN genes. This current is activated on hyperpolarization and deactivates very slowly. Therefore, a depolarizing sag develops during hyperpolarization that depolarizes a cell toward resting potential and causes an afterdepolarization following a hyperpolarizing event.¹⁰⁸ Under appropriate conditions, such currents cause bursting in subicular neurons and also in CA1 and CA3 neurons. These currents, together with M -type potassium currents and persistent sodium currents, are frequently involved in membrane potential oscillations, which occur already at rest or close to action potential threshold.⁷⁰^,⁷²^,¹¹² These membrane potential oscillations have been observed in neocortical neurons and are readily induced in perirhinal and entorhinal cortex cells.¹²⁸ They also occur in certain interneurons.¹¹³ These types of ionic currents also induce resonance behavior.⁴⁵^,¹²⁹ In cells that express these currents, the input resistance changes as a function of frequency of current input to the neurons. Resonant cells frequently have the largest impedance at frequencies between 4 and 15 Hz. This implies that these cells integrate synaptic input more effectively when it occurs at resonance frequencies. Such mechanisms are important in determining oscillatory activity preceding seizures, contribute to θ and γ activity, and are also involved in α rhythm activity. It is expected that such mechanisms are altered in epilepsy.

Voltage-Gated Currents and Epilepsy

There is considerable evidence that voltage-gated currents contribute to the generation of seizure. This evidence stems from various observations. It has been shown that toxins that prolong Na⁺ channel opening cause seizures.⁵¹ Similarly, drugs that prevent activation of K⁺ currents also induce seizures. These include muscarinic receptor agonists, which reduce M-type currents, certain Na-dependent K conductances and EAG channel activity, and 4-AP, which can block K⁺ currents. Surprisingly, TEA, which affects many K⁺ currents, is a much less potent convulsant agent than 4-AP. Because A-type currents are rapidly inactivated during depolarization, it is unlikely that they contribute much to epileptogenesis. When TEA is applied in a concentration of 2 mM, which reduces I_K in a similar way as 4-AP at a concentration of 50 to 100 μM, short recurrent discharges with a frequency of 0.1 Hz are induced in entorhinal cortex and hippocampal slices, whereas 4-AP induces convulsion-like events. Because 4-AP augments presynaptic uptake of Ca²⁺ much more than TEA, it is likely that augmented transmitter release causes the seizures.⁵⁴^,⁷⁸ The second line of evidence comes from experiments with transgenic mice. Many of the potassium-channel-knockout animals develop epilepsy. Mutations in ion channels have also been found in inherited epilepsies in mice and rats. These studies are often also supported by studies of human inherited epilepsies in which Na channel mutations, Ca channel mutations, and K channel mutations have all been identified as causes of epilepsy.

It is of interest that epilepsies can also be accompanied by acquired channelopathies. Thus, it was shown in a number of convincing studies that bursting behavior of neurons is upregulated in different acquired epilepsies.¹⁴² The best-studied channels in this respect are HCN channels, which are upregulated following hyperthermia-induced seizures.²⁷^,¹³⁰ Upregulation of these channels has also been seen after repetitive stimulation of a kind that induces neither long-term potentiation nor long-term depression, and it was suggested that they are involved in homeostatic plasticity.¹⁵¹ Changes in expression of K currents have been described in a model of temporal lobe epilepsy in which downregulation of the A-type-encoding K_V4.2 channel has been described.¹² This channel is involved in limiting backpropagation of action potentials and in reducing the transfer of excitatory postsynaptic potential (EPSPs) from apical dendrites into the soma. It is interesting that in the same model upregulation of T-type Ca channels also has been described, resulting in increased bursting behavior of hippocampal neurons.¹⁴² For acutely isolated hippocampal neurons of the CA1 region, it was shown that the amplitude of voltage-gated Ca²⁺ currents of the HVA type is increased after kindling.⁸⁸ Enhancement of Ca²⁺ currents persisted for at least 6 weeks without further tetanic stimulation. Whole-cell patch-clamp recordings of acutely dissociated granule cells from the dentate gyrus of kindled adult rats demonstrated a markedly enhanced Ca²⁺-dependent inactivation of Ca²⁺ currents, which was correlated with lack of the cytoplasmic Ca²⁺-binding protein calbindin-D_28K during kindling-induced epilepsy.⁸⁷^,⁸⁸

Many of the voltage-gated currents are targets for antiepileptic drugs. The effects on Na⁺ currents of some of the antiepileptic drugs, such as phenytoin and carbamazepine, are well known.⁹⁵^,¹²⁰ Agents that act on presynaptic uptake of Ca²⁺,³⁹ or that activate K⁺ currents, are of considerable interest. Thus, it has been shown that drugs that activate ATP-dependent K⁺ currents, M currents, and Ca-activated K currents can have antiepileptic effects.⁷^,⁹^,¹⁰^,⁵³ More recently, some interest has developed in the properties of Na⁺ currents in epileptic tissue of patients with drug-resistant epilepsy. It may well be that transient Na⁺ currents in the tissue of such patients have a reduced sensitivity to phenytoin and carbamazepine.¹¹⁷

Ligand-Gated Ionic Channels

Ligand-gated ionic channels open when a neurotransmitter binds to a receptor. These channels also comprise various subunits. Ligand-gated channels open fast, in the range of milliseconds, and are therefore used in fast synaptic transmission. The most important inhibitory ligands are GABA and glycine; they open a Cl^-- (and HCO₃^--) permeable channel.⁷⁹ Depending on membrane potential and the reversal potential for Cl^-, this results in either depolarization or hyperpolarization. The Cl^- reversal potential depends on the activity of a Cl^- pump.⁹⁴ If this is inwardly directed, mediated by the NKCC1 transporter, the Cl^- equilibrium potential is above the resting membrane potential, and consequently a depolarizing GABA or glycine potential results. When the Cl^- transporter is outward mediated by the KCC2 transporter, the Cl^- potential is hyperpolarizing with respect to the resting membrane potential, and hyperpolarizing inhibitory postsynaptic potentials are generated on binding of the agonist to the receptor. In addition to NKCC1 and KCC2, the carbanhydrase activity also plays a role, which determines the bicarbonate reversal potential.¹¹⁹^,¹⁵²

Depending on subunit composition, the GABA-operated ion channel is also permeable to bicarbonate. Because the bicarbonate equilibrium potential is depolarizing with respect to

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resting membrane potential, bicarbonate-permeable ion channels exert a depolarizing drive, and the resulting GABA-mediated potentials are depolarizing. In fact, after an initial hyperpolarization, prolonged activation of GABA channels lead to a depolarization that is sensitive to carbanhydrase inhibitors. The subunit composition also determines whether GABA-gated channels respond to furosemide, zinc, benzodiazepines, and β-carbolines. In general, barbiturates seem to prolong the mean open time of GABA-gated Cl^- channels (see Chapter 23). A subgroup of GABA receptors does not fit the pharmacologic sensitivity of GABA receptors. These receptors contain ρ subunits. Their role in synaptic mechanisms is not fully understood.

GABA receptors containing δ subunits¹³⁷ have a high affinity for GABA and therefore sense ambient GABA levels. These receptors mediate tonic inhibition.¹³⁴ Recent evidence suggests that tonic inhibition is very sensitive to neurosteroids.⁹⁷ It is likely that tonic inhibition is affected in epilepsy.⁵⁶^,⁸⁵^,¹³¹

A potentially important second mechanism providing for tonic inhibition relies on activity-dependent editing of glycine receptors with a subsequent manifold increase of glycine receptors to glycine, making them sensitive to ambient levels of glycine and taurine.⁹⁹ Preliminary evidence suggests that this occurs also in human temporal lobe epilepsy.

Acetylcholine, when bound to nicotinergic receptors, and glutamate, when bound to AMPA, kainate, or NMDA receptors, are ionic channels permeable to small cations such as Na⁺ and K⁺ and to a variable degree also to Ca²⁺ ions (see Chapter 22). These ionic channels mediate fast excitatory synaptic transmission. In addition, serotonin can bind to receptors that gate an ion channel directly. The 5HT3 receptors are predominantly expressed on interneurons. Nicotinergic receptors are also important, however, as shown by the recent discovery that a mutation in an α subunit of the nicotinergic receptor accounts for a genetically determined type of epilepsy in humans.⁸⁵^,¹³⁶

For excitatory synaptic transmission in the central nervous system, the glutamate receptors are most important. The duration of excitatory postsynaptic potentials in the central nervous system is largely regulated by a fast desensitization,⁴²^,¹¹⁴ whereas the duration of inhibitory postsynaptic potentials is regulated by GABA uptake into presynaptic terminals and glia.⁴¹^,⁴⁴

Metabotropic Receptors

Receptors are said to be metabotropic when the binding of a ligand does not directly lead to a conformational change of an ion channel. Instead, the receptors interact with G proteins. These can directly influence membrane channels, particularly Ca²⁺ channels,¹²¹ or the activation of enzymes can lead to the production of second messengers such as inositol-1,4,5-triphosphate (IP3), cyclic adenosine monophosphate (cAMP), and diacylglycerol, which activate protein kinases (see Chapter 27). These affect voltage- and ligand-gated channels in the membrane. The effects are rather slow and therefore account for slow depolarizations or hyperpolarizations. Activation of metabotropic receptors often produces long-lasting effects, in that phosphorylation of an ion channel persists until the channel is used; the information conveyed by an appropriate stimulus also affects later synaptic potentials and the integration properties of neurons for subsequent signals. Finally, second messengers can activate gene-regulating peptides and thereby affect the production of proteins, a process involved in the formation of long-lasting memory traces.

Three classes of ionotropic transmitters (GABA, glutamate, and acetylcholine) interact with metabotropic receptors. Whereas changes in metabotropic GABA_B receptors and mGluR receptors have been well studied in different epilepsy models, less is known on the alterations in muscarinergic receptors and the cholinergic signaling pathway. Neuromodulators such as serotonin, norepinephrine, dopamine, and histamine also interact with metabotropic receptors. Finally, most neuroactive peptides act via G proteins.

The activation of metabotropic receptors leads to different cellular effects that can be either excitatory or inhibitory. This depends on the type of G protein activated, the intracellular second messenger, and the target in a given cell as well as in a given neuronal network. Thus, glutamate can probably activate three different classes of metabotropic receptors.³⁷^,⁹⁸ Potassium channels are activated by GABA via GABA_B receptors, but GABA also inhibits Ca²⁺ currents. Acetylcholine and glutamate often block K⁺ channels via metabotropic receptors. The same applies to serotonin (5-HT) and norepinephrine. These agents, however, can also activate K⁺ currents. In general, metabotropic receptors are likely future targets for anticonvulsant drugs.¹⁰¹

Presynaptic Receptors

As initially demonstrated in the spinal cord, it is now well established that presynaptic terminals express both ionotropic and metabotropic receptors. These can be autoreceptors, in which the released transmitter binds to the presynaptic membrane and then interferes with further transmitter release. They can also be heteroreceptors, in which the released transmitter diffuses to terminals in the surrounding area. In the spinal cord and brainstem, presynaptic terminals are themselves innervated by afferent fibers. There, ionotropic GABA receptors can impose a depolarization on the axon terminal, which blocks Na⁺ currents through depolarization, thereby interfering with synaptic transmission.¹²⁶ In cortical structures, inhibitory effects of GABA_B receptors on presynaptic GABA release have been frequently demonstrated.³⁶^,¹⁴⁷ Glutamatergic nerve endings also possess presynaptic autoreceptors, however, which often differ from postsynaptic receptors in their binding properties, including those for kainate¹²⁷ and mGluR receptors.¹²³ Presynaptic receptors modulating transmitter release have been described for adenosine and many neuromodulators and neuropeptides as well. Their physiologic and pathophysiologic function is little understood. However, such presynaptic receptors may be suitable targets for new classes of antiepileptic drugs (FIGURE 4).

FIGURE 4. Location of different presynaptic and postsynaptic transmitter receptors. Receptors for γ-aminobutyric acid B (GABA_B), adenosine, and glutamate can regulate transmitter release. AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; NMDA, N-methyl-D-aspartic acid; R, receptor.

Summary and Conclusions

The trafficking of information in the central nervous system depends critically on voltage- and transmitter-gated ionic channels. Storage of information and adaptation to metabolic needs probably depend on metabotropic receptors, which process short-term information into longer-lasting modifications of neuronal transfer of information and may even alter the preferred state of neuronal firing. Through activation of gene-regulatory peptides, they appear to be involved in long-term storage of information of physiologic or pathologic significance.

Any transfer of information leads to a perturbation of ionic gradients, which requires the functioning of ion transporters. Thus, ion pumps are involved not only in regulating membrane potential and loading the “battery” for activation of voltage- and transmitter-gated currents, but also in setting up ionic gradients for the uptake of nutrients and the outward transport of metabolites. Ion transporters are usually not electrically neutral and so contribute to the electrical behavior of neurons and glia.

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Through modern techniques of molecular biology, information about the binding properties of voltage- and transmitter-gated channels is expanding, and new approaches to the development of region-specific toxins and drugs are being undertaken. These will soon offer more-specific tools for manipulating neuronal activity. Increased knowledge of the presynaptic terminal, with its autoreceptors and heteroreceptors and various amino acid transporters, offers new possibilities for the development of antiepileptic drugs. Better understanding of the role of metabotropic receptors and their individual functions may even make it possible for drugs to be designed that interfere with the epileptogenic process itself.

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	Epilepsy: A Comprehensive Textbook 2nd Edition
	© 2008 Lippincott Williams & Wilkins

Epilepsy: A Comprehensive Textbook2nd Edition

Epilepsy: A Comprehensive Textbook
2nd Edition