10.3: Diffusion Across a Membrane - Channels

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Pentameric Ligand-Gated Ion Channels (pLGICs)

• Describe the structural organization of pentameric ligand-gated ion channels, distinguish between orthosteric and allosteric binding sites, and explain how ligand binding at the extracellular domain is transduced through TM2/TM3 rearrangements to gate ion flow, using the nicotinic acetylcholine receptor and GLIC as specific examples.
• Predict the physiological consequence — excitation or inhibition of a neuron — of opening a cation-selective versus anion-selective pLGIC, connecting the direction of ion flow to changes in transmembrane potential (depolarization vs. hyperpolarization) given the typical ion gradients across a mammalian neuronal membrane.

Voltage-Gated Ion Channels

• Explain the structural basis of K⁺ selectivity in voltage-gated K⁺ channels (Kv), describing how the conserved TTVGYG selectivity filter in the P-loop discriminates K⁺ from the smaller Na⁺ ion through dehydration energetics and geometric constraints, and how the "knock-on" mechanism facilitates rapid vectorial ion flow through the pore.
• Describe how the S4 voltage-sensor helix — with its repeating positively charged Arg and Lys residues — detects changes in transmembrane potential and transduces this signal through coupled conformational changes to open or close the activation gate, and explain the "ball-and-chain" mechanism of Kv channel inactivation and its regulation by inner-leaflet PIP₂.
• Compare the structural organization of voltage-gated K⁺ channels (homotetramers of six-helix subunits) with voltage-gated Na⁺ channels (single polypeptide with four pseudo-symmetric domains I-IV), and explain how the three-state gating model — closed, open, and fast-inactivated — controls the timing and duration of Na⁺ influx during an action potential, with reference to the IFM motif and its intracellular receptor.
• Explain how the DEKA selectivity filter in eukaryotic Na⁺ channels confers Na⁺ selectivity over K⁺ and Ca²⁺, and describe the pharmacological significance of the multiple neurotoxin binding sites (sites 1–6) in NaV channels, using tetrodotoxin (pore blocker) and α-scorpion toxin (fast-inactivation inhibitor) as contrasting examples.

Lipid-Gated and Mechanosensitive Ion Channels

• Explain how the inner-leaflet lipid PIP₂ acts as an agonist for the inward rectifier potassium channel Kir2.2, describe how PIP₂ binding between the transmembrane and intracellular domains produces a conformational change that gates K⁺ influx, and explain how this inward K⁺ current — despite flowing against the concentration gradient — is thermodynamically driven by the transmembrane electrical potential and block of outward flow by polyamines and Mg²⁺.
• Describe the gating mechanism of the mechanosensitive TRAAK K⁺ channel, explaining how physical deformation of the bilayer displaces an occluding lipid from the cytoplasmic pore entrance, and connect this to the broader principle that membrane protein function can be regulated by the physical and chemical state of the surrounding lipid bilayer.

Introduction

If you punched a hole or pore in the membrane, depending on its size, multiple types of chemical species could flow through it simultaneously. We'll talk about pores in the next section. Let's focus on channels with much smaller openings that are gated open to allow ion flow through them. They are often called ionotropic receptors. Channels can be "gated" open by many mechanisms, including ligand binding, change in membrane potential, lipid interactions, and mechanical stress. Opening a channel to ion flow allows rapid transmission of information (in this case, an electrical signal) into the cell, leading to rapid cellular responses. This is an ideal signaling mechanism for neural cells, which demand quick responses.

We'll show examples of each type of gating mechanism. Before we do, it is helpful to know typical extracellular and intracellular ion concentrations in a mammalian neuron, for example (Table \(\PageIndex{1}\)).

Table \(\PageIndex{1}\): Extracellular and intracellular ion concentration in a mammalian neuron

ion	extracellular = [ion]out (mM)	intracellular = [ion]in (mM)
Na^+	145 mM	5-10 mM
K^+	5 mM	140 mM
Cl^-	110 mM	10 mM
Ca^2^+ (free)	1.2 mM	100 nM

When ion channels are opened in neural cell membranes, the direction of favorable thermodynamic flow is down a concentration (chemical potential) gradient. However, the direction is also affected by the transmembrane potential. Typical resting potentials of neural cells are about -60 to -70 mV (negative inside). When a nonspecific cation channel is gated open, the kinetic barriers to diffusion are relieved. At that moment, Na⁺ ions would flow in due to both the chemical and electrical potential.  K⁺ ions would flow out, but with a lower driving force because the negative transmembrane potential hinders their efflux. How are such large gradients of these ions formed? We'll answer that in the next section on active transport.

Pentameric ligand-gated ion channels (pLGICs)

These channels play a key role in neuronal signaling. They are ligand-gated channels. In neural systems, the ligands are neurotransmitters. All are comprised of five monomers, forming the functional channel together with the pore formed in the center of the pentameric structure. The subunits can be identical (homopentamer) or different (heteropentamer). All have "Cys-loop" motifs, so they have also been called Cys-loop receptors. Examples include the mammalian nicotinic acetylcholine, serotonin (5-HT), γ-aminobutyric (GABA), glycine, and glutamate receptors.

Figure \(\PageIndex{1}\) shows the generic structure of the pLGICs

Figure \(\PageIndex{1}\): Generic structures of pentameric ligand-gated ion channels. Smelt, Charles L.C.; (2019) Allosteric modulation of pentameric ligand-gated ion channels. Doctoral thesis (Ph.D.), UCL (University College London). https://discovery.ucl.ac.uk/id/eprint/10075893/. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/ 4.0/)

The monomeric structure is shown on the left. Each contains four transmembrane helices (TM1-4). A top-down view of the pentameric structure is shown to the right. The pore surface forms at the interface of the central TM2 helices. The ligand (neurotransmitter) binds to the extracellular domain with contributions from all the subunits. On ligand binding, TM2 and TM3 rearrange to allow the formation of a transient pore and passive diffusion of specific ions.

pLGICs are incredibly interesting and pharmacologically relevant. In general, they have two different types of binding sites.

Orthosteric sites bind ligands in the extracellular domains. A conformational change rearranges helices, opening the pore when bound. The natural ligand is also called the agonist, as it promotes the ion channel's function (either neuron excitation or inhibition). The natural ligand/agonist binding opens the channel to ion flow. This can lead to the activation or excitation of the neural cell if positive ions flow into the cell, depolarizing it as the transmembrane potential becomes more positive. Neurotransmitters that lead to this response are excitatory. Alternatively, binding of inhibitory neurotransmitters at the orthosteric site can inhibit neural cell activation if the channel is a ligand-gated anion channel. This hyperpolarizes (makes the transmembrane potential more negative), thereby inhibiting neural cell activation. Inhibitors or antagonists of channel function, whose structure typically resembles at least somewhat the structure of the endogenous ligand or agonist, also bind to the orthosteric site.

Allosteric sites are distal to the orthosteric site. Ligands that bind to allosteric sites also lead to conformational changes that either augment or diminish the effect of normal ligand/agonist binding by modulating ion flow through the pore.

pLGICs interact with analgesics and anesthetics, which makes them even more interesting.

Figure \(\PageIndex{2}\) shows two excitatory pLGICs, the 5-hydroxytryptamine (5HT) (left) and nicotinic acetylcholine (right) receptors. The ligand binds in an orthosteric site in the extracellular domain (ECD), which is composed mostly of beta secondary structure. Allosteric sites are often found in the transmembrane domain.

Figure \(\PageIndex{2}\): Two excitatory pLGICs, the 5-hydroxytryptamine (5HT) (left) and nicotinic acetylcholine (right) receptors. Adapted from Lara César O., Burgos Carlos F., Moraga-Cid Gustavo, Carrasco Mónica A., Yévenes Gonzalo E. Pentameric Ligand-Gated Ion Channels as Pharmacological Targets Against Chronic Pain, Frontiers in Pharmacology, 11 (2020), 167. https://www.frontiersin.org/article/...har.2020.00167, Creative Commons Attribution License (CC BY).

Likewise, Figure \(\PageIndex{3}\) shows orthosteric binding sites for GABA and glycine, as well as the binding of modulators that can bind in the TMD or, in the case of benzodiazepines, in the ECD as well.

Figure \(\PageIndex{3}\): Orthosteric binding sites for GABA and glycine, adapted from Lara César O., Burgos Carlos F., Moraga-Cid Gustavo, Carrasco Mónica A., Yévenes Gonzalo E. Pentameric Ligand-Gated Ion Channels as Pharmacological Targets Against Chronic Pain, Frontiers in Pharmacology, 11 (2020), 167. https://www.frontiersin.org/article/...har.2020.00167, Creative Commons Attribution License (CC BY).

 

How do inhalational anesthetics work?

We have used them for decades, but their mechanism of action remains unclear!

Details!

The mechanisms by which anesthetics work are still unclear. These fascinating molecules can alter function in various organisms, including bacteria, yeast, worms, flies, plants, and animals. Of course, their effect on consciousness appears only in animals. Their selective "turning off" of a function (consciousness) of an entire organ (the brain) is stunning! One theory suggests that they exert their effects through bulk changes in the lipid bilayer, as the potency of anesthetics is generally related to their hydrophobicity. Most are nonpolar and have long been known to work on membranes, presumably altering ion flow through neural membrane ion channels. Typical inhalational and intravenous anesthetics are shown in Figure \(\PageIndex{3}\), along with their date of first use.

Figure \(\PageIndex{3}\): Structure of common inhalational and intravenous anesthetics. Adapted from Eur J Anaesthesiol. 2009 Oct; 26(10): 807–820. doi: 10.1097/EJA.0b013e32832d6b0f

A strong correlation is observed between the minimum alveolar concentration (MAC, in atmospheres) of inhalational anesthetics and their partition coefficient into olive oil (a measure of their hydrophobicity). This is illustrated in the Meyer-Overton plot shown in Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): The Meyer-Overton plot of minimum alveolar concentrations (in atmospheres) vs partition coefficient into olive oil for inhalation anesthetics. https://commons.wikimedia.org/wiki/F...orrelation.png

The MAC is the concentration of inhaled anesthetic within the alveoli at which 50% of people do not move in response to a surgical stimulus (i.e., it is much like an IC₅₀ for receptor inhibition). What's so fascinating is the range of molecular species that can act as anesthetics, including N2 and most Noble gases. Consider N₂, a nonpolar and nonreactive molecule, which comprises 80% of the atmosphere. From the graph, it is evident that high pressure is required for it to exert its anesthetic properties. That can occur when scuba diving using regular air in tanks. Divers can experience nitrogen narcosis (also called depth intoxication or rapture of the deep) when using just compressed air. Hence, a mixture of 21% oxygen, 35% helium, and 44% nitrogen is often used. Dentists use nitric oxide to alter consciousness and pain perception, but it does not cause general anesthesia except in some who can be hypersensitive to its use.

Additional studies suggest protein:anesthetic interactions are important. For example, their activity affects the water-soluble protein luciferase. Some molecules (like dichlorohexafluorocyclobutane), expected to have anesthetic properties based on their hydrophobicity, don't. The (S) enantiomer of isoflurane is 50% more potent than the (R) enantiomer in rats, which is hard to explain based on nonspecific partitioning into a bilayer.

Most modern theories suggest that they more directly affect specific target proteins and their proximal interacting lipids in neuromembrane bilayers. The main targets of anesthetics appear to be pLGICs. Anesthetics reduce neuron excitability and firing. Hence, you could hypothesize that they inhibit excitatory pLGICs (such as the 5HT and acetylcholine receptors) and/or activate inhibitory ones, such as the GABA and glycine receptors. pLGICs are pharmacological targets of many general anesthetics. However, anesthetic inhibition of certain GABA channels and potentiation of nicotinic acetylcholine channels have also been observed.

Recent elegant studies have shown that the inhaled anesthetics chloroform and isoflurane affect K⁺ ion flow through the potassium channel subfamily K member 2, also known as the outward rectifying potassium channel protein TREK-1. The channel switches between a voltage-insensitive potassium leak channel and a phosphorylated voltage-dependent outward-rectifying potassium channel. It doesn't affect the channel protein directly but does so indirectly by altering the local membrane, which, in turn, affects the localization of phospholipase D₂ (PLD₂), a membrane-anchored protein covalently modified with palmitic acid. PLD2 hydrolyzes phosphatidylcholine, with a positively charged choline head group, to choline and phosphatidic acid, with a negatively charged phosphate head group

The effect of these general anesthetics appears to be on lipid rafts in neural membranes. Lipid rafts are enriched in cholesterol and saturated lipids, especially sphingomyelins such as monosialotetrahexosylganglioside1 [GM1]. Rafts are especially important in the brain, where cholesterol can reach up to 45% of plasma membrane lipids. Typical rafts are about 100 nm in diameter. In the presence of hydrophobic anesthetics, rafts become larger and more dispersed as the anesthetics partition into them.

Membrane proteins also partition into rafts. One such protein is phospholipase D2 (PLD2), which is targeted to the inner leaflet rafts by post-translational palmitoylation. In the presence of general anesthetics, PLD1 laterally translates away from the disrupted and enlarged lipid raft and binds to the disordered C-terminal region of TREK-1. This localizes PLD2 and helps activate it, producing high local concentrations of phosphatidic acid, which has a negatively charged head group. That group interacts with a positive region of the TREK-1 protein, inducing a conformational change that opens the channel to K⁺ efflux. In effect, PLD2 activates TREK-1 through the local formation of phosphatidic acids.

The opening of the channel hyperpolarizes the cell membrane (making the inside more negative and inhibiting neural activity, a hallmark of anesthesia). These concerted actions are shown in Figure \(\PageIndex{a}\) below.

Figure \(\PageIndex{a}\): Effects of general https://www.pnas.org/content/117/24/13757. r Creative Commons Attribution-NonCommercialNoDerivatives License 4.0 (CC BY-NC-ND)

Deletion of the TREK-1 gene reduces the anesthetic effect. If a catalytically inactive PLD2 mutant (K758R) is overexpressed, all effects of chloroform are eliminated.

We will explore two pLGIC, the eukaryotic nicotinic acetylcholine channel and a prokaryotic analog, GLIC.

Nicotinic acetylcholine channel (6cnj)

One very interesting channel is the one involved in nicotine addiction. It binds nicotine (an exogenous alkaloid) and the normal endogenous neurotransmitter, acetylcholine. Both compete for the same orthosteric binding site. Since the binding of nicotine gates the channel open, nicotine acts as an agonist. The similarities in their structures are illustrated in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Structures of nicotine and acetylcholine

The membrane protein is a ligand (acetylcholine)-gated (open-close) positive ion (Na⁺ or K⁺) channel involved in fast neural communication (such as at the neuromuscular junction). The quaternary structure of the pentameric receptor consists of two α4 and three β2 subunits - (α4)₂(β2)₃. This isoform is the most abundant in the human brain and the one involved in nicotine addiction.

The iCn3D model (6CNJ) below has two bound nicotines (spacefill) in the extracellular domain and one Na⁺ ion (spacefill) in the transmembrane domain containing the pore. Na⁺ or K⁺ ions flow down a concentration gradient across the membrane, driven by a thermodynamically favored process.

Figure \(\PageIndex{5}\) shows an interactive iCn3D model of the nicotinic acetylcholine channel with two bound nicotines (spacefill) in the extracellular domain and one Na⁺ ion (spacefill) in the transmembrane domain containing the pore (6CNJ). (long load time)

Figure \(\PageIndex{5}\): Nicotinic acetylcholine channel with two bound nicotines (spacefill) in the extracellular domain and one Na⁺ ion (spacefill) in the transmembrane domain containing the pore (6CNJ). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...rbXmpaha6FYNB8

The gold, blue, and brown β2 subunits are glycosylated on Asn 143 and are shown with a Man (β4) GlcNAc (β4) GlcNAc N-linked oligosaccharide. Nicotine is bound between two alpha-beta interfaces. One is shown between the green (alpha) and gold (beta) subunits, and the other is between the magenta (alpha) and blue (beta) subunits.

GLIC: A prokaryotic pLGIC

This protein is a proton-gated cation channel that is specific for Na⁺ and K⁺, which diffuse down their electrochemical gradients. In a sense, H⁺s in the extracellular side act as "ligands" as the channel is opened with increasing H⁺ concentration (decreasing pH) on the outside of prokaryotic cells. The protein is homologous to eukaryotic pLGICs. Structures of the protein from Gloeobacter violaceus bound to propofol, an anesthetic, are known. GLICs also interact with ethanol and barbiturates. Hence, they serve as models to elucidate the binding and effects of anesthetics.

In contrast to eukaryotic pLGICs, the "ligand - H⁺" does not bind in the orthosteric site in the extracellular domain occupied by traditional ligands. Rather, changes in protonation states of key proton acceptors and donors in the protein lead to conformational changes analogous to those found on binding ligands to orthosteric sites on classical pLGICs. The external pH associated with half-maximal inward current, pH₅₀, is approximately 5.1 ± 0.2.

Evidence suggests that when pH is lowered from 7 to 4, Glu 35 (distant from the orthosteric site), with a pKa ≈ 5.8, becomes protonated. It connects to other H⁺ acceptors and donors in the open form via a hydrogen-bond network. These include two triads of amino acids found at the interface between the extracellular (ECD) and transmembrane (TMD) domains. R192-D122-D32 comprises a conserved "electrostatic triad." The second is Y197-Y119-K248. The network allows bridging of the effects, starting with Glu 35 in the ECD into the transmembrane region, where allosteric effectors usually interact with the protein.

Figure \(\PageIndex{6}\) shows an interactive iCn3D model of the open form of GLIC (3P50) with bound propofol (long load time)

Figure \(\PageIndex{6}\): The open form of the prokaryotic pentameric GLIC ion channel with bound propofol (3P50). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...v92cdeunVY65R8 (long load time)

Orient the iCn3D model below with the extracellular domain (mostly beta structure) at the top and the transmembrane domain (alpha-helical) at the bottom. Key molecular players involved in the interactions described above, from the top down:

• Glu 35 (stick, color CPK)
• R192-D122-D32 electrostatic triad (sphere, CPK color)
• Y197-Y119-K248 triad (stick, color magenta)
• propofol (sphere, color CPK)

Propofol and another anesthetic, desflurane, bind at the same site localized in the upper part of the transmembrane domain of each of the five subunits.

The model below shows the mostly nonpolar (induced dipole-induced dipole) interactions between a bound propofol and side chains in the TMD. Also shown is an interaction between phosphatidylcholine and propofol.

Figure \(\PageIndex{7}\) shows an interactive iCn3D model showing the mostly nonpolar (induced dipole-induced dipole) interactions between one bound propofol and side chains in the TMD. Also shown is an interaction between phosphatidylcholine and propofol. (long load time)

Figure \(\PageIndex{7}\): Propofol interactions in the transmembrane domain of the pentameric GLIC ion channel (3P50). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...s8dJnmX7SFKjLA

Voltage-Gated Ion Channels

In contrast to pentameric ligand-gated ion channels, which require five monomeric subunits to aggregate into a quaternary structure to form a pentameric pore, voltage-gated ion channels can form a channel from an aggregate of monomeric proteins, each containing a single six-transmembrane helical unit, or from a longer polypeptide containing multiple repeating 6-transmembrane helical units.

Figure \(\PageIndex{8}\) shows a cartoon of a common voltage-gated K⁺ channel. The monomer (top), denoted as the α subunit, contains a transmembrane domain containing six helix segments. Four of these monomers aggregate to form the actual homo- or heterotetrameric channel (bottom).

Figure \(\PageIndex{8}\): Cartoon of a common voltage-gated K⁺ channel

The genes for the K_v channel family, which facilitate K⁺ diffusion across the membrane, encode α subunits of approximately 500 amino acids and a molecular weight of about 57,000. Four of these α subunits come together in the membrane to form the functional channel, a tetramer of α subunits, forming a single central pore together. The α subunit can form homo- or heterotetramers with different α-subunit-encoding gene products. In addition, the functional channel has smaller regulatory β-subunits.

A cartoon structure of a typical voltage-gated Na⁺ channel is shown in Figure \(\PageIndex{9}\). It is a single polypeptide (α) chain that contains four sequential repeats of the six transmembrane helical segments (I-IV). The functional channel (bottom) has just one polypeptide chain.

Figure \(\PageIndex{9}\): Cartoon structure of a typical voltage-gated Na⁺ channel adapted from https://www.anaesthesiajournal.co.uk...264-4/fulltext

The Na⁺ channel has a molecular weight of around 229 kDa and about 2000 amino acids (each 4x that of the K⁺ channel α subunit). It is glycosylated and subjected to multiple post-translational modifications. Usually, the protein in the central nervous system is a complex of the α subunit and small additional regulatory β subunits, which modify the kinetics and voltage-dependency of the α subunit channel.

Segment (helix) 4 of each of the four repeat units illustrated above is the conserved "voltage" sensor. It contains multiple charged amino acids whose disposition changes with changes in the transmembrane potential, allowing conformational changes in the protein and gating of ion flow. Each of the four repeating units above also contains an extracellular P-loop (colored purple in segment I) connecting helix 5 and helix 6.

We will focus on K⁺ and Na⁺ channels below.

K⁺ Permeation through Kv1.2 Channel

Voltage-dependent potassium channels (Kv) have four subunits and can be homo- or heterotetramers. They allow the voltage-gated flow of potassium ions through the membrane. Several obvious questions should arise. How can they be selective for K⁺ ions? How can they allow the larger K⁺ ions to flow passively through and not the smaller Na⁺ ions? Secondly, how can a change in the transmembrane potential open or close the channel? That question boils down to how changes in transmembrane potential can alter protein conformation. We'll show several iCn3D models of this protein.

Figure \(\PageIndex{10}\) shows a simplified view of the rat Kv1.2 channel (3lut) from top and side views (without parts of the cytoplasmic domain), showing each of the four identical monomeric subunits in a different color. Each monomer has S1-S6 transmembrane segments.

https://www.ncbi.nlm.nih.gov/Structu...cn3d/full.html

Figure \(\PageIndex{10}\): Rat Kv1.2 channel (3lut) from top and side views (without parts of the cytoplasmic domain), showing each of the 4 identical monomeric subunits in a different color.

The protein is found in the central and peripheral nervous systems and cardiovascular systems. K⁺ ions diffuse through the center from the extracellular to intracellular side down a concentration gradient but potentially against the transmembrane potential. The four monomers in the homotetramer form one central pore.

Figure \(\PageIndex{11}\) shows an interactive iCn3D model showing the rat Kv1.2 channel (3lut) described in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{11}\): Rat Kv1.2 channel (3lut) . (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...99VbNxVzCZyr9A

Figure \(\PageIndex{12}\) shows an interactive iCn3D model showing the S4 voltage sensor helix in each monomer of the Kv1.2 potassium channel (3lut) .

Figure \(\PageIndex{12}\): S4 voltage sensor helix in each monomer of the Kv1.2 potassium channel (3lut) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...nZnNTpqSwA6Ab9

Figure \(\PageIndex{13}\) shows a more detailed structure of the Kv1.2 potassium channel (3lut). The four monomers, which pack together to form the tetramer, are shown in light and dark grey to allow key residues highlighted in color to stand out. The details are explained below.

Figure \(\PageIndex{13}\): Detailed structure of the Kv1.2 potassium channel (3lut)

The left images show the channel from the side (top-left view) and the top (bottom-left view). The right images show only one monomer, with different side chains highlighted.

Figure \(\PageIndex{14}\) shows an interactive iCn3D model detailing key residues in the workings of the Kv1.2 potassium channel (3lut)

Figure \(\PageIndex{14}\): Key residues in the workings of the Kv1.2 potassium channel (3lut) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...wYWMWmHWp1UP6A

K⁺ selectivity - Even if not voltage-gated, all potassium ion channels solve the selectivity dilemma similarly. All have, at the narrowest part of the pore in the center of the channel, this consensus sequence - Thr-Thr-Val-Gly-Try-Gly (TTVGYG) - which is found in the P-loop. These are shown in gold and brown colors in the figure above. The -OHs in the selectivity filter can interact with a dehydrated K ion but not with a dehydrated Na ion, which can not approach close enough to form significant interactions. Surrounding the filter are twelve aromatic amino acids, which constrain the size of the pore opening. The interactions of the filter O's with the K ion compensate for the energetically unfavorable dehydration of the ion. The filter contains K⁺ ions, which repel one another, facilitating the vectorial discharge of ions through the membrane. These ions must form weak interactions with the selectivity filter. The pore is mostly hydrophobic, facilitating the flow of ions through the membrane.

Figure \(\PageIndex{15}\): below shows a close-up of the selectivity filter. Four Thr 374s (second Thr in the selectivity filter sequence of TTVGYG) from the four different monomers in the channel are clearly shown interacting with the top K⁺ ion (gray sphere).

Figure \(\PageIndex{15}\): K⁺ Selectivity filter of the Kv1.2 potassium channel (3lut)

Figure \(\PageIndex{16}\) shows an interactive iCn3D model detailing key residues in the K⁺ selectivity filter of the Kv1.2 potassium channel (3lut). Hover over the residues to identify them.

Figure \(\PageIndex{16}\): Key residues in residues in the K⁺ selectivity filter of the Kv1.2 potassium channel (3lut) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...FFKJVVHtbpuYj6

Different voltage-gated ion channels alter ion selectivity through changes in these amino acids in the P-loop, as illustrated below in Table \(\PageIndex{2}\). As channels lose their specificity for K⁺, they gain specificity for Na⁺ and Ca²⁺. Red highlights denote conserved residues, and yellow highlights denote residues that are chemically similar.

sequence	specificity
TVGYG	strong K^+ channels
CIGYG	weak K^+, HCN channels
TVGDG	TRP channels
STFEG	ionotropic glutamate receptors
LCGEW	strong Ca^2^+ voltage-gated channels

Table \(\PageIndex{2}\): P-loop specificity side chains in voltage-gated ion channels

Voltage gating -

The Helix S4 in each monomer of the complex's transmembrane domain is the voltage sensor. The sequence of this helix is LAILRVIRLVRVFRIFKLSRH. Note that the arginines and lysines are highlighted in blue. They repeat every three amino acids. The voltage-sensor domain must be shielded from the nonpolar acyl of the bilayer. Four conserved Arg residues on S4, part of the voltage-sensor domains, are shielded from the lipids and coupled to an amphiphilic helix running parallel to the plane of the membrane. The arginines move under the influence of forces arising from changes in the membrane's electric field, initiated by ion movement through other ion channels. The electric field does mechanical work on the voltage sensor as charged Arg residues are moved through it. The movement is coupled through the amphiphilic helix to the pore, which undergoes conformational changes. In turn, the voltage sensor's S4 and coupled S5 helices perform mechanical work on the pore by altering its conformation to open/close it, specifically at the pore's activation gate. This seems quite similar to how iron movement into the heme plane in hemoglobin, upon oxygenation, pulls the proximal His on the F8 helix, which then transmits a conformational change to other helices in the subunit, leading to cooperative conformational changes in the tightly packed protein. About 12 charges move across the transmembrane potential field.

Channels, once open, must be inactivated. In the voltage-gated potassium channel, inactivation occurs when the amino-terminal cytoplasmic domain binds to the potassium pore on the cytoplasmic side, in an interaction likened to a "ball on a chain" (the cytoplasmic domain) binding to the pore opening. The chain tethers the ball domain, allowing it to swing and bind to the pore opening. The ball domain contains both positively charged and hydrophobic regions. Where is the ball domain in the absence of inhibition? Recent studies (Oliver et al.) have shown that a positive domain can bind to phosphatidylinositol 4,5-bisphosphate (PIP2) lipids in the inner leaflet of the membrane bilayer. When so bound, the channel cannot be deactivated. As you will see in the next section, PIP2 can also be cleaved to form diacylglycerol and inositol 1,4,5-trisphosphate when cells are activated by external factors (hormones, growth factors, etc) in the process of signal transduction.

Figure \(\PageIndex{17}\) shows a molecular dynamics simulation showing K⁺ interaction with the channel lining and the "knock-on" mechanism showing how an incoming K⁺ ion can repel a K⁺ ion in the pore through the channel.

Figure \(\PageIndex{17}\): Molecular dynamics simulation of K⁺ movement through a channel

 

Voltage-gated sodium channels (Na_V)

The eukaryotic voltage-gated sodium channels (NaV) allow the inward movement of Na+ ions, which depolarize neurons and trigger an action potential in nerve and muscle cells. (For more information on neuron signaling, see Chapter 28.9).   The NaV has an alpha subunit that forms the pore, plus beta subunits that associate with it and modulate its activity. Nine eukaryotic isoforms exist.  NaV has four domains, I-IV,  each containing segments 1-6. Each of the S1-S4 segments forms a voltage-sensitive domain, and each of the S5 and S6s forms the pore.  In contrast to eukaryotic NaVs, which have a single chain, bacterial NaVs contain four identical subunits.  Post-translational modification of the alpha subunit can regulate its activity.  

As important as it is to initiate Na⁺ influx to trigger neuron firing, it is equally important to turn it off to control neural signaling.  The inward Na⁺ current is rapidly inactivated within a few milliseconds. 

Many neurotoxins bind to NaV channels and regulate their activity.  Key examples are the α-scorpion and sea anemone toxins, which both inhibit the fast inactivation of the NaV, leading to prolonged or sequential action potentials.  These toxins bind to the voltage sensor in domain IV, which is key for the fast inactivation in the absence of the toxin. 

The S4 helical segments in each domain are the key voltage sensors.  Each S4 segment contains 4-6 positively charged Arg and Lys side chains. On depolarization of the cell (when the inside becomes less negative and more positive), this helix moves "up" from the cytoplasmic side (which is increasingly more positive), which opens a voltage-gated channel.   Specificity for the small Na⁺ ion (over the K⁺ and Ca²⁺ ions) is determined mainly by four amino acids, DEKA (Asp-400, Glu-755, Lys-1237, and Ala-1529) found in P loops of domain I-IV, respectively. This selectivity filter is conserved.        

Figure \(\PageIndex{18}\) below shows a cartoon of the NaV with two associated regulatory beta chains.

Figure \(\PageIndex{18}\): Cartoon of the eukaryotic voltage-gated sodium channel (Nav) with two associated regulatory beta chains.   https://www.guidetopharmacology.org/...rd?familyId=82. CC BY-SA 4.0

Note the sites for posttranslational modification by phosphorylation and drug interactions.  Cylinders represent probable α-helical segments S1-S6. Bold lines represent the polypeptide chains of the selectivity filter and tetrodotoxin binding site. The yellow S4 segments are the voltage sensors.  The "h" in the blue circle is in the inactivation gate loop.  Blue circles are sites implicated in forming the inactivation gate receptor. Sites of binding of α- and β-scorpion toxins (ScTX) and a site of interaction between α and β1 subunits are also shown. Tetrodotoxin is a specific blocker of the sodium channel pore. In contrast, the α- and β-scorpion toxins block fast inactivation and enhance activation, respectively, generating a persistent sodium current that causes depolarization block of nerve conduction.

Table \(\PageIndex{3}\) shows the different types of neurotoxin receptor sites found in NaVs

Neurotoxin Receptor Site #	Toxin or Drug	Domains
1	Tetrodotoxin	IS2–S6, IIS2–S6
	Saxitoxin	IIIS2–S6, IVS2–S6
	µ-Conotoxin
2	Veratridine	IS6, IVS6
	Batrachotoxin
	Grayanotoxin
3	α-Scorpion toxins	IS5–IS6, IVS3–S4
	Sea anemone toxins	IVS5–S6
4	β-Scorpion toxins	IIS1–S2, IIS3–S4
5	Brevetoxins	IS6, IVS5
	Ciguatoxins
6	δ-Conotoxins	IVS3–S4
local anesthetic drug sites	Local anesthetic drugs	IS6, IIIS6, IVS6
	Antiarrhythmic drugs
	Antiepileptic drugs

Table \(\PageIndex{3}\): Neurotoxin receptor sites in NaVs.  https://www.guidetopharmacology.org/...rd?familyId=82. CC BY-SA 4.0

Figure \(\PageIndex{19}\) shows a view of the effective pore in the bacterial Nav.

Figure \(\PageIndex{19}\): Structure of the bacterial sodium channel NavAb pore B. Architecture of the NavAb pore. Glu177 side chains in the P loop are shown in purple.  The pore volume is shown in grey. The P and P2 alpha helices that form the scaffold for the selectivity filter and outer vestibule are shown in green and red, respectively. https://www.guidetopharmacology.org/...rd?familyId=82. CC BY-SA 4.0

As mentioned above, the bacteria NaVs have four monomeric subunits.  In contrast to the K⁺ channel, which requires K⁺ ions to be dehydrated to form sufficient interactions with the pore and to pass through, the Na⁺ ions need to be hydrated. Figure \(\PageIndex{20}\) shows an interactive iCn3D model of the A and B chains of the bacterial NaV voltage-gated sodium channel pore and C-terminal domain (5BZB)

Figure \(\PageIndex{20}\):  A and B chains of  bacterial NavMs voltage-gated sodium channel pore and C-terminal domain (5BZB)
(Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...vRmDqmDHGsYLh8

One subunit is shown in a transparent cyan surface, and the second in magenta.  Three Na⁺ ions are shown (gray spheres).  Eight water molecules are shown interacting with them in the pore.  The other two subunits are not shown for clarity.

The voltage-gated sodium channel has three major conformational states:

• a basal closed state found at resting cell potentials, in which an activation gate occludes the pore for Na⁺
• an open state found when a depolarizing potential is reached in the cell
• an inactivated state formed within 10 ms of opening of the channel when the inactivation gate with a a Ile-Phe-Met (IFM) sequence motif, found in the intracellular linker between domain III and IV (near the cytoplasmic face of the receptor) closes off the pore to further Na⁺ entry. 

The protein converts back to the closed state when the transmembrane potential is restored to its initial value (around -70 mV), and the positively charged S4 segments move back towards the cytoplasmic face.

A cartoon of the three-state model for the Na⁺ channels (as other voltage-gated ion channels in general) is shown in Figure \(\PageIndex{21}\) below.

Figure \(\PageIndex{21}\): Three-state model for the voltage-gated ion channels.  Hinard, Valerie & Britan, A & Rougier, Jean-Sébastien & Bairoch, Amos & Abriel, Hugues & Gaudet, Pascale. (2016). ICEPO: The ion channel electrophysiology ontology. Database: the journal of biological databases and curation. 2016. 10.1093/database/baw017. Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/)

The YouTube video below by Pete Meighan provides an incredibly clear description of the three conformational states of the channel and the transitions from closed to open to inactivated.

 

To understand these conformational states, we need to look at the protein structure in greater detail.  Figure \(\PageIndex{22}\) below shows multiple representations of the voltage-gated sodium channel. The pore domain PD is formed from helix segments 4 and 5 on each of the IV domains of the protein. The voltage sensor domain is formed from the S1-S4 segments, of which segment S4, containing multiple positively-charged Arg and Lys, is key.

Figure \(\PageIndex{22}\): Structure features of the voltage-gated sodium channel.  Dongol, Y.; C. Cardoso, F.; Lewis, R.J. Spider Knottin Pharmacology at Voltage-Gated Sodium Channels and Their Potential to Modulate Pain Pathways. Toxins 2019, 11, 626. https://doi.org/10.3390/toxins11110626.  Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

Panel (A) shows a schematic representation of the α-subunit of the voltage-gated sodium (Na_V) channel. Four non-identical domains (DI–DIV) feature six neurotoxin receptor sites (Sites 1–6) and key residues that contribute to the outer Na⁺ ion selectivity filter (EEDD) and the inner selectivity filter (DEKA). The connecting S5–S6 linker, called the P-loop (P), together with the S5 and S6 segments from each domain, contributes to the formation of the Na⁺ ion-selective channel pore.  Sites 1-6 (colored purple, green, cyan, magenta, etc.) are sites where inhibitors, such as toxins, bind.

Panel (B) shows the structure of the Na_V1.7 channel (PDB 6J8G). Four voltage-sensing domains (VSDs), DI (yellow), DII (blue), DIII (green), and DIV (orange), are shown with their corresponding pore-forming segments (S5 and S6) arranged to form the pore domain (PD) selective to Na⁺ ions. The P-loop that contributes to forming the inner selectivity filter is colored in red spheres (DEKA), and the outer selectivity filter (EEDD) is colored in purple. The S6 segments of all four domains contribute to forming the intracellular pore region. Site 3 (cyan) and Site 4 (pink) are the major binding sites for spider knottins (neurotoxins). The β1 and β2 subunits that interact with DIII and DI, respectively, are highlighted in beige color.

Panel (C) shows a schematic of the protein's three main conformational states that control the NaV channel gating. At polarized potentials, the DI–DIV S4 segments are drawn towards the intracellular side due to the positive gating charges to render the closed conformation (down state). Depolarization relieves the forces holding the down state, and DI–DIII S4 segments are rapidly released extracellularly to open the S6 channel gate in the open conformation (up state). Note the movement of the S4 helix with its positive charge toward the extracellular side of the membrane. The DIV S4 moves up slowly compared to the DI–DIII S4 and drives fast inactivation, during which the channel is intracellularly occluded by the Ile, Phe, and Met (IFM) motif. After cell repolarization, the channel returns to a closed (resting) state.

Figure \(\PageIndex{23}\) shows an interactive iCn3D model of a ternary complex of human Nav1.2 with the beta2 regulatory subunit and conotoxin IIIA (6J8E)

Figure \(\PageIndex{23}\): Human sodium channel Nav1.2-beta2-KIIIA ternary complex (6J8E)
(Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?aYxomdUqZHqbMmE5A

Domains I-IV are gray, yellow, green, and cyan, respectively. The separate beta-2 regulatory subunit is shown in magenta. The positive side chains in each S4 segment of the four domains are shown as red sticks.  The side chains of the inner four amino acids (DEKA) comprising the selectivity filter are shown in spacefill, with CPK colors and labels.  The brown peptide cartoon on the extracellular side (red sphere layer for outer leaflet) is the µ-conotoxin KIIIA.  The spacefill molecule in the pore near the blue sphere layer representing the inner leaflet is the neurotoxin veratridine (VTD), which inhibits channel inactivation and lengthens the action potential with possibly fatal consequences.  A single Na⁺ ion is shown at the top of the pore as an orange sphere labeled Na.  The µ-conotoxin blocks the pore.  The Ile1488-Phe1489-Met1490 (IFM) motif, found in the intracellular linker between domains III and IV (near the cytoplasmic face of the receptor) and responsible for fast inactivation, is shown in gray spheres and labeled with single-letter codes. 

The selectivity filter of DEKA differs from that of another sodium channel (NavAb), which has four glutamates.  Asp and Ala line the wall of the filter region, and Glu and Lys can attract/release the Na⁺ ion. The IFM motif must interact with an"inactivation gate receptor" within the protein for fast inactivation. Short intracellular loops connecting all the S4 and S5 segments are likely candidates for this.  The inactivation gate has three key amino acids, F1651, L1660, and N1662.

Figure \(\PageIndex{24}\) shows another interactive iCn3D model of a ternary complex of human Nav1.2 with the beta2 regulatory subunit and conotoxin IIIA (6J8E) highlighting just the DEKA selectivity filter, the IFM motif, and its receptor inactivation gate F1651, L1660 and N1662. 

Figure \(\PageIndex{24}\): Human sodium channel Nav1.2-beta2-KIIIA ternary complex highlighting just the DEKA selectivity filter and the IFM and its receptor inactivation  gate F1651, L1660 and N1662 (6J8E).  (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?tq3kXbT44GeCCUcY7

They are all shown in spacefill and labeled.  Orient the molecule with the cytoplasmic domain at the top.  The bilayer leaflets are omitted for clarity, but the cytoplasmic pore inhibitor is still shown in sticks.

Figure \(\PageIndex{25}\) below shows a close-up of the bound Na⁺ ion.

Figure \(\PageIndex{25}\): Sodium ion in the SF of the PDB ID 6J8E Na_v1.2 cryo-EM structure. The pore blocker μ-conotoxin KIIIA is not shown. Alberini et al. J. Chem. Theory Comput. 2023, 19, 10, 2953–2972. April 28, 2023. https://doi.org/10.1021/acs.jctc.2c00990. Creative Commons.

Structures of the closed, open, and inactivated states of NaV1.5 are now known.  Key regions are shown in Figure \(\PageIndex{26}\) below.  The open state was the hardest to solve since it closes in milliseconds to form the inactive state, as described above.  Mutations in the Ile-Phe-Met (IFM) motif to QQQ prevented inactivation.  This would ordinarily be deleterious to an organism, but in the presence of the small-molecule inhibitor propafenone, it was possible to isolate this state. 

Figure \(\PageIndex{26}\):  Closed, open, and inactivated conformations of the activation gate and the locations of arrhythmia mutations.  Daohua Jiang, Richard Banh, Tamer M. Gamal El-Din, Lige Tonggu, Michael J. Lenaeus, Régis Pomès, Ning Zheng, William A. Catterall.  Open-state structure and pore gating mechanism of the cardiac sodium channel.  Cell, Volume 184, Issue 20, 2021, 5151-5162.e11, ISSN 0092-8674,. https://doi.org/10.1016/j.cell.2021.08.021. Reprinted with permission from Elsevier.  May not be sublicensed, assigned, or transferred to any other person without the publisher's written permission.

Panel (A) shows the closed activation gate of Na_V1.5 generated by MODELER based on the resting-state structure of Na_VAb (PDB: 6P6W), sealed by a square of hydrophobic side chains of hydrophobic residues V413, L941, I1471, and I1773 (spacefill, black) that in the closed state completely seal off the cytoplasmic opening in the pore. These rings of amino acid side chains come together on conformational changes resulting from the engagement of the IFM motif with its internal receptor.

Panel (B) shows the open activation gate of Na_V1.5/QQQ triple mutation. Red arrows indicate the directions of movement of S6 segments compared to the resting state.

Panel (C) shows the partially open but nonconductive activation gate of rNa_V1.5_C in the inactivated state. Red arrows indicate the directions of movement of the S6 segments compared to the open state.

Panels (D–F) show the structures from (A) to (C) in a space-filling surface representation, with hydrated Na⁺ placed in the central cavity behind the activation gate. Red and green spheres represent water and Na⁺, respectively. van der Waals distances measured across the orifice of the activation gate are 4.3 Å (DI-DIII) × 2.8 Å (DII-DIV) for the resting/closed state, 6.9 Å (DI-DIII) × 5.0 Å (DII-DIV) for the inactivated state, and 7.3 Å (DI-DIII) × 8.2 Å (DII-DIV) for the Na_V1.5/QQQ open-state structure.

Panels (G–I) show structures from (A) to (C) with the locations of arrhythmia mutations causing LQT-3 overlaid as green spheres.

The spacefill models in D-E clearly show that the cytoplasmic opening is "open" in the open state E (obviously) and occluded to increasing degrees in the inactive state (F) and closed state D. 

Finally, figure \(\PageIndex{27}\) shows an interactive iCn3D model of the electrostatic surface potential of the rat Na_v1.5 channel (6UZ3)

Figure \(\PageIndex{27}\): Electrostatic surface potential of the rat Na_v1.5 channel (6UZ3)
(Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...UZdQeUUPtYGgR6

The red indicates negative potential, and the blue indicates positive potential.  The white is neutral.  Note that the bilayer representations are essentially neutral with respect to their effect on the overall surface potential. Tilt the model to view the top and bottom entrances to the pore and the overall negative charge density expected in the net attraction of the positive Na⁺ ions.

Lipid-Gated Ion Channels

Membrane receptors are embedded in the lipid bilayer, so it should not be surprising that specific lipids might bind and trigger conformational changes in the receptor, thereby mediating specific biological activity. The specific lipid might form during an upstream event and then bind to the receptor, triggering a signal transduction pathway, which we will explore in the next chapter. Look at receptors gated by binding the lipid phosphatidylinositol bisphosphate (PIP₂). It is found in small concentrations in membranes and can be cleaved in by phospholipase C to form a small polar intracellular signaling molecule, IP₃ (discussed in Chapter 12). PIP₂ can also recruit proteins to the membrane and participate in signaling events. In this section, we will discuss how it binds to a K⁺ ion channel protein and opens, allowing K₊ to flow into cells (in the opposite direction of the normal efflux), which helps control the transmembrane potential.

Kir2.2 -Inward rectifier potassium channel Kir2.2

The names of this channel can best be understood by understanding the meaning of the word 'rectify'. The verb has several meanings, but for us, the best definition is to correct. What does this channel correct? When is it functional? We'll explore the details more in the next chapter, but first, we need a better understanding of what happens to the actual Na⁺ and K⁺ ion concentrations outside and inside the cell during neural activation. How much do they change? We need this to understand the driving force for the Kir2.2 channels that move K⁺ into the cell, which is in the opposite direction of the usual flow. In that sense, it is "rectifying" the K⁺ concentrations.

How many ions actually move on opening a channel?

It's less than you think!

Click for the details!

When a neural cell is activated or fires, the cell membrane potential goes from a resting potential of around -60 to -70 mV (inside negative ) to a more positive potential as Na⁺ ions enter the cell through voltage-gated Na⁺ channels (after an early neurotransmitter-gated ion channel is opened after ligand binding) with the ions flowing down a chemical and electric potential gradient. At a certain membrane potential (about +30 mV), K⁺ channels open, allowing an efflux of K⁺, down both a chemical and an electric potential gradient, returning the cell potential close to its equilibrium value of around -60 to -70 mV (inside negative). But how much do the actual K⁺ and Na⁺ ion concentrations change in this process? The somewhat counterintuitive answer is hardly any at all!

We need to understand first how the membrane acts as a capacitor. The charge Q on the surface of a plate or side of a membrane is proportional to the voltage across the plate or membrane. Figure \(\PageIndex{28}\) shows how a membrane with a transmembrane potential acts as a capacitor. The dielectric medium in the capacitor determines how quickly the charges on the plates dissipate. When the medium is an insulator resistant to charge flow, the plates remain charged longer. The same is true for the membrane. The hydrophobic bilayer acts as an insulator, resisting the discharge of the membrane potential. The bilayer offers high resistance (low conductance) to the flow of charge. Only when channels are open, and the ions become reasonably permeable to flow, does the membrane potential change over short periods. The following derivation is adapted.

Figure \(\PageIndex{28}\): A membrane as a capacitor. After https://openpress.usask.ca/physics15...d-capacitance/. License: CC BY: Attribution

It makes sense that the stored charge (Q) on either side of the membrane is proportional to the membrane voltage. We can write the following simple equations:

\begin{equation}
\begin{array}{l}
Q \propto V \\
Q=\mathrm{C} V
\end{array}
\end{equation}

where C, the proportionality constant, is the capacitance with units of the Faraday (which you remember from introductory chemistry). Let's normalize this equation for an area of 1 cm². The measured capacitance of lipid bilayers is about 10^-6F/cm². Let's assume a voltage change from -70 mV to + 30 mV for a total of 0.1 V. Hence

\begin{equation}
Q=\frac{10^{-6} \mathrm{~F}}{\mathrm{~cm}^{2}}(0.1 \mathrm{~V})=\frac{10^{-7} \mathrm{Coul}}{\mathrm{cm}^{2}}
\end{equation}

Let's convert that to the number of elementary charges on the membrane surface per μm2.

\begin{equation}
\left(\frac{10^{-7} \mathrm{Coul}}{1 \mathrm{~cm}^{2}}\right) x\left(\frac{1 \text { ion }}{1.6 \times 10^{-19} \mathrm{Coul}}\right)=\frac{6.25 \times 10^{11} \text { ions }}{\mathrm{cm}^{2}} \times \frac{1 \mathrm{~cm}^{2}}{10^{8} \mu \mathrm{m}^{2}}=\frac{6,250 \text { ions }}{\mu \mathrm{m}^{2}}
\end{equation}

What does this mean for an ordinary eukaryotic cell? Let's model the cell as a sphere with a diameter of 10 μm.

Knowing the equations for the volume (V = (4/3)πr³) and surface area (4πr²), the volume of a 10 μm cell is about 524 μm³, and the surface area is 314 μm². The table below shows the cell's resting ion concentrations and the number of ions in the 524 μm³ volume of the cell (column 3).

Using the calculated value of 6250 ions moved/μm², the total number of K⁺ and, accordingly, Na⁺ ions that move across 314 μm² of total cell membrane surface area is about 2 million. The results are shown in Table \(\PageIndex{3}\) below.

1. ion	2. [ion]intracellular (mM)	3. # ionsintracellular	4. total ions moved during neuron response	5. change in [ion]intracellular
sodium	10 mM	3.2 × 10^9 Na^+	~ 2,000,000 in on depolarization	~6.3 x 10^-2 mM (~0.6% change)
potassium	150 mM	4.7 × 10^10K^+	~ 2,000,000 out on repolarization	~6.3 x 10^-2 mM (~0.6% change)

Table \(\PageIndex{3}\): How many ions move across a membrane

\begin{equation}
\frac{2 \times 10^{6} \mathrm{Na}^{+} x\left(\frac{1 \mathrm{~mol} \mathrm{Na}^{+}}{6.022 \times 10^{23} \mathrm{Na}^{+}}\right)}{524 \mu M^{3} \times\left(\frac{1 \mathrm{~L}}{10^{15} \mu M^{3}}\right)}=6.3 \times 10^{-6} \mathrm{M}=6.3 \times 10^{-3} \mathrm{mM}
\end{equation}

The actual change in intracellular Na⁺ ion concentration on excitation is only about 0.6% of the initial [Na⁺]_{intracellular} concentration on excitation and depolarization of the cell. We can also assume that the K⁺ ion changes to the same degree in repolarization. So when the Na⁺ and K⁺ channels open, the "flood gates" are not opened. Permeability increases, but this results in a small influx of Na⁺ ions, insufficient to significantly change the intracellular Na⁺ concentration. It is, however, enough to change the transmembrane potential! It's a misconception that there are significant changes in ion concentration across the membrane on depolarization and repolarization of the cell. The Goldman-Hodgkin-Katz equation (previous section) shows that the membrane potential depends on both concentrations and permeability coefficients.

Now, we can present the inward rectifying potassium channels, which facilitate the movement of K⁺ into the cell from the outside. This is in the opposite direction of the usual flow of the ions. Since the ions are moving towards higher levels of K⁺ inside the cell, it would appear to be an example of either passive diffusion, favored only by the electrical potential, or active transport. None of these is true. K⁺ ions diffuse from outside to inside the cell since outward diffusion is substantially blocked by molecules such as polyamines and Mg²⁺! We don't have to invoke active transport or a violation of the basic rules of thermodynamics. The channels thus display strong inward currents and weak outward ones.

The channels allow large K⁺ ion conduction when the membrane potential is more negative than the resting K+ ion equilibrium potential, but less when it is more positive. Hence, the net effect is to maintain the resting K⁺ potential.

There are several subfamilies of the Kir channel. They are always potentially active (open) except those gated by G-proteins (see next chapter) or by ATP binding. (These are involved in metabolism.) Phospholipids and proteins regulate Kir activity. Now, we will explore the Kir 2.2 channel, which is gated open by PIP₂, a membrane lipid, rather thanan agonist of the Kir2.2 by changes in the transmembrane potential. Yet by opening this channel and allowing the inward flow of K⁺ ions, PIP₂ regulates the transmembrane potential. It is the agonist for the Kir 2.2 channel.

Instead of having monomeric units with S1-S6 transmembrane helical segments with a voltage sensor (S4), P loop, and S5-S6 selectivity filter, it has just two transmembrane helices. The N- and C-termini are both in the cytoplasm, connected by an extracellular loop (H5), which helps form the prototypical K⁺ selectivity filter with the consensus sequence T-X-G-Y(F)-G. Similar to the voltage-gated K⁺ channel, four of these aggregate to form homo- or heterotetramers in the membrane. Figure \(\PageIndex{29}\) illustrates these points.

Figure \(\PageIndex{29}\): The Kir potassium channel

The proteins have large intracellular domains (ICD). On binding of PIP₂ to the region between the transmembrane and ICD, which produces a large conformational change, allowing K⁺influx. The model below shows the R186A mutant tetrameric Kir 2.2 channel (3SPG) with four bound PIP₂ analogs containing two short fatty acids (octanol) esterified to the glycerol backbone. There appear to be two lipid-binding sites: a nonspecific site in the TMD and a specific site for PIP₂ in the ICD.

Figure \(\PageIndex{30}\) shows an interactive iCn3D model of the inward rectifying R186A mutant tetrameric Kir 2.2 channel (3SPG) with four bound PIP₂ analogs containing two short fatty acids (octanol) esterified to the glycerol backbone. Hover over the residues to identify them.

Figure \(\PageIndex{30}\): Inward rectifying R186A mutant tetrameric Kir 2.2 channel (3SPG). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3...S5vuBsdbSSqwV8 (long load time)

Figure \(\PageIndex{31}\) shows an animation that shows the monomeric Kir protein morphing from the apo state (without PIP₂, 3JYC) to the PIP₂-bound state (3SPI).

Figure \(\PageIndex{31}\): Monomeric Kir protein morphing from the apo state (without PIP₂, 3JYC) to the PIP₂-bound state (3SPI)

Another PIP₂-gated ion channel is the transient receptor protein (TRP) channel. These channels play a role in vascular tone. Smooth muscle cells express TRPC3 and TRPC6 channels, as well as Na⁺ or Ca²⁺ channels that depolarize, leading to smooth muscle contraction and vasoconstriction.

Mechanosensitive channels

We saw in the previous chapter that some pores (not channels) are gated open not by voltage, specific agonists such as neurotransmitters, or even specific lipids like PIP₂, but by more general changes in membrane bilayer properties (membrane tension, curvature, pressure). Likewise, specific channel proteins can be gated by changes in the bilayer's physical properties. We will consider one mechanically-active channel, TRAAK.

TRAAK - Potassium channel subfamily K member 4

The protein is a K⁺ ion channel that is not voltage-sensitive but opens in response to mechanical deformation of the bilayer. The channel can be opened by making the cytoplasm basic and raising the temperature. It is involved in pain sensation and pressure transduction. The TRAAK 4 opening is occluded by lipids from the inner leaflet. If physical changes remove the lipid, transmembrane helix 4 rotates, preventing the lipid from blocking the channel and opening it. Further changes in the coupled transmembrane helices 2 and 3 stabilize the opening.

Figure \(\PageIndex{32}\) shows an interactive iCn3D model of the TRAAK channel protein (4wff) in the closed state. Hover over the residues to identify them.

Figure \(\PageIndex{32}\): Potassium channel subfamily K member 4 TRAAK channel protein in the closed (4wff) state. Hover over the residues to identify them. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...UzX6p5d36VicdA

The K⁺ ions are aligned in the channel. Decane, a nonpolar molecule, is shown in spacefill and colored cyan. The decane is probably decanoic acid, and the carboxyl group was not defined in the structure due to its high flexibility. This suggests that the "decanoic acid" is not tightly bound. It binds through the cytoplasmic side through an opening in the membrane protein. In the open state, transmembrane helix 4 rotates, blocking access to the cavity. Hence, lipid binding closes the channel.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter examines the structural and mechanistic diversity of ion channels — gated membrane pores that allow rapid, selective, and thermodynamically driven ion flow — focusing on the four major gating mechanisms: ligand binding, voltage sensing, lipid binding, and mechanical deformation.

Ion channels are distinguished from carrier proteins by their mode of operation: rather than translocating individual solutes through repeated conformational cycles, channels form a continuous hydrophilic pathway that, once opened, allows millions of ions per second to flow passively down their electrochemical gradients. The direction and magnitude of ion flow are governed by the transmembrane potential and the ion concentration gradients established by active transport. In a resting neuron, Na⁺ is concentrated outside (~145 mM) and K⁺ inside (~140 mM); the resting potential is approximately -60 to -70 mV (negative inside). When a nonselective cation channel opens, Na⁺ floods inward driven by both its concentration gradient and the electrical potential, depolarizing the cell.

Pentameric ligand-gated ion channels (pLGICs) are the molecular basis of fast synaptic transmission. Each functional channel is a pentamer of subunits, each contributing four transmembrane helices, with the central TM2 helices from all five subunits lining the ion-conducting pore. Neurotransmitters (agonists) bind orthosteric sites in the extracellular domain, triggering rearrangements of TM2 and TM3 that transiently open the pore. Cation-selective pLGICs — such as the nicotinic acetylcholine receptor — depolarize the postsynaptic cell and are excitatory, while anion-selective pLGICs — such as the GABA_A and glycine receptors — hyperpolarize the cell and are inhibitory. Allosteric modulators binding within the transmembrane domain can augment or diminish agonist efficacy without directly competing for the orthosteric site. The prokaryotic homolog GLIC, gated by extracellular acidification rather than a conventional neurotransmitter, serves as a structural model for understanding how conformational changes propagate from the extracellular domain to the pore, and for dissecting the binding sites of general anesthetics such as propofol, which bind within the transmembrane domain through predominantly hydrophobic interactions.

Voltage-gated ion channels are gated not by ligands but by changes in the transmembrane electric field. Voltage-gated K⁺ channels (Kv) are homotetramers of six-helix subunits (S1-S6), with the S5-S6 segments and intervening P-loop from each subunit forming the central pore. The conserved TTVGYG sequence in the P-loop constitutes the selectivity filter: its backbone carbonyl oxygens precisely mimic the hydration shell of K⁺, allowing the dehydrated ion to pass through efficiently, while the smaller Na⁺ ion cannot approach close enough to make equivalent interactions, making dehydration energetically prohibitive. Multiple K⁺ ions occupy the filter simultaneously, and electrostatic repulsion between adjacent ions — the "knock-on" mechanism — drives rapid vectorial flux. The S4 helix, bearing multiple Arg and Lys residues spaced every three positions, acts as the voltage sensor: membrane depolarization exerts an electrostatic force on these charges, driving S4 outward and propagating conformational changes through the amphiphilic linker helix to open the activation gate at the cytoplasmic end of S6. Channel inactivation occurs when the cytoplasmic N-terminal "ball" domain occludes the open pore, with PIP2 on the inner leaflet competing for the ball domain and thereby modulating the rate of inactivation.

Voltage-gated Na⁺ channels (NaV) share the same voltage-sensing and gating logic but are organized as a single polypeptide containing four pseudo-symmetric domains (I-IV), each with six transmembrane segments. Na⁺ selectivity is conferred by the DEKA filter (Asp-Glu-Lys-Ala from domains I-IV, respectively), which accommodates the smaller, hydrated Na⁺ ion. The channel cycles through three states: a closed resting state at negative potentials, a transiently open state upon depolarization, and a fast-inactivated state entered within milliseconds when the cytoplasmic IFM motif (Ile-Phe-Met) of the domain III-IV linker occludes the pore by binding to an intracellular receptor formed by short linker sequences. The kinetic asymmetry between the rapid opening of domains I-III and the slower movement of the domain IV voltage sensor drives the coupling between activation and fast inactivation. NaV channels contain at least six pharmacologically distinct toxin-binding sites; tetrodotoxin blocks the pore directly, while α-scorpion toxins impair domain IV movement and thereby prolong Na⁺ entry with potentially fatal depolarizing consequences.

Beyond ligand- and voltage-gating, ion channels can be regulated by specific lipids and mechanical forces. The inward rectifier K⁺ channel Kir2.2 is opened by PIP2, a minor inner-leaflet phosphoinositide, which binds at the junction of the two-helix transmembrane domain and the large cytoplasmic domain, driving a conformational change that gates K⁺ influx. Although K⁺ influx is directed against the concentration gradient, it is thermodynamically driven by the membrane potential and facilitated by the polyamine and Mg²⁺ block of outward K⁺ flow. The net effect of Kir2.2 activity is to stabilize the resting membrane potential. The mechanosensitive TRAAK channel illustrates yet another gating mechanism: physical deformation of the bilayer (from pressure, temperature, or changes in membrane tension) displaces a lipid molecule that normally occludes the cytoplasmic channel entrance, allowing transmembrane helix 4 to rotate and stabilize the open state. This example reinforces the chapter's closing theme — and the broader course principle — that the physical state and lipid composition of the bilayer are not merely a passive backdrop for membrane protein function, but active regulators of channel gating and ion flux.