Stabilization of poorly populated ion channels’ states by the modified bilayer content

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Short history of ion channels’ discovery: from the experiments of Galvani to the first crystal structure

Our encounter with ion channels began in the XVIII century with the first experiments of Galvani on frogs: a frog preparation, consisting of the lower body half with exposed nerves and a metal wire inserted across the vertebral canal, started contracting when Galvani touched its nerves with a lancet, and a spark rose from a distant electric machine [7]. Galvani concluded that the animal tissue contained an innate vital force, which he termed “animal electricity”. Since that time, the history of ion channels’ discovery remained intimately connected with the study of a nerve impulse’s initiation and propagation.
The processes of initiation and propagation of a nerve impulse are mediated by functioning of specific ion channels, called voltage-gated, sensitive to changes in the TM potential. These channels constitute only one class among all the diversity of ion channels characterized so far. Below, we will briefly discuss the modern classification of ion channels and show, which place in it voltage-gated ion channels occupy. This Section is mostly based on the review of Bezanilla [8] and on the book of Ochs [9].

Toward the work of Hodgkin and Huxley

Since the experiments of Galvani, the next major breakthrough in the ion channels’ field, which was made by Nobel laureates Hodjkin and Huxley, had to wait two centuries. During this time, several important concepts were developed that prepared the scientific community for understanding of initiation and propagation of a nerve impulse in terms of ions passing through specialized proteins in the plasma membrane.
• At the end of the XIX century – the beginning of the XX century two of these concepts were formulated. The first one postulates the existence of self-consistent charged species, or ions: acids and bases are substances that dissociate in water, yielding electrically charged atoms or molecules (Arrhenius theory of dissociation). The second concept states for a lipoid nature of the barrier surrounding living cells: there is a positive correlation between lipid solubility of the compound and its anesthetic potency (Meyer and Overton lipid solubility-anesthetic potency correlation).
• Another important observation was made by Nobili and Matteucci [9]. They found a potential difference in muscle between a site that had been cut or injured and an intact part of the muscle, a phenomenon they termed the “injury potential” or “demarcation potential”. The potential difference so produced causes a flow of current, the “injury current”, between the two sites of the muscle. This observation led to a hypothesis that, during initiation and propagation of a nerve impulse, the resting potential of the membrane changes to an action one (see 2.2.1).
• At the beginning of the XX century, Bernstein has postulated that the resting potential is created due to selective permeability of the membrane to different ionic species [9]: while potassium ions diffuse following their electrochemical gradient, others are not able to permeate. The potential so produced is given by the Nernst equation (W. Nernst was awarded a Nobel Prize in Chemistry, 1920): = ![ ]! + ! »[ ]! + ! »[ ]! . ( ![ ]! + !![ ]! + ! »[ ]!.
Here, ! is the relative permeability of an ion , where the latter substitutes for potassium, sodium or chloride; [ ]! and [ ]! denote concentrations of an ion inside the cell and at the extracellular solution respectively; is the gas constant, is the temperature, and is the Faraday constant. The action potential was further attributed to a transient increase of ionic current through the membrane. Along with the evolving concepts of the resting and action membrane potentials, development of a new experimental technique, allowing one to record directly ionic current flowing across the membrane without any resultant change in the membrane potential, played an important role in the work of Hodgkin and Huxley. This technique, called voltage-clamp [2], was first proposed by Cole and was further improved by Hodgkin and Huxley. Briefly, the setup (see Figure uses two electrodes: one to record the membrane voltage (V2), and the other to apply current to the cell (V2 or I2). The command potential is maintained by a negative feedback system. The electrodes are connected to an amplifier (amp), measuring the membrane potential and translating the signal to a feedback amplifier (clamping amp). The latter subtracts the membrane potential from the command one and sends an output signal to the electrode (V2 or I2). If the membrane potential deviates from the command one, the appropriate electrode (V2 or I2) conducts a current into the cell, reducing this deviation to zero. The injected current is monitored via a current-to-voltage converter to provide a measure of the total membrane current.

Overall architecture of an ion channel captured from the voltage clamp experiments

After the seminal work of Hodgkin and Huxley, it became widely accepted that ions pass across the membrane through discrete sites, namely, ion channels. However, the nature of these channels was still debated: whether they are holes in the membrane or highly specialized proteins embedded into it. Rojas and Luxoro provided the first evidence advocating for the proteic nature of ion channels [26]. They demonstrated that a specific protease (pronase) is able to destroy inactivation of sodium current completely, though leaving potassium current unaltered. Another piece of evidence came from Agnew and colleagues [27]. They extracted a 230 kDa protein from an eel electric organ; embedded into liposomes or bilayers, this protein was shown to produce sodium current [28,29]. The first insight into the architecture of ion channels came from the experiments with toxins that temporary block ionic currents. Hence, applying tetraethylammonium (TEA) and its derivatives to a giant axon, Armstrong found that the block of potassium current occurred after the channel opening
[30]. This observation led to the concept of a discrete gate. This gate was further attributed to the intracellular region of the channel, while the extracellular region was proposed to be responsible of ionic selectivity [31]. In 1959, Mullins wrote that ions entering the channel loose their hydration shell at least partially, thus allowing the channel to distinguish between different types of ions [32]. Before his work, selectivity of the membrane was assumed to stem from the difference in radius of hydrated potassium and sodium ions. However, Mullins noticed that rubidium and cesium ions have hydrated radii very close to that of potassium, but they behave quite differently with respect to their ability to depolarize the membrane or to penetrate though it. “… This difficulty can be avoided by assuming that ions move though pores that fit them rather closely; these pores serve to replace the hydration that the ion would have in an aqueous solution …”.

Patch clamp, an advanced technique for measuring ionic currents

While the voltage clamp was a widely used technique for measuring ionic currents, it could only be applied to rather big cells, as sharp microelectrodes were needed to penetrate the membrane. In the late 1970s, Sakmann and Neher suggested an alternative technique, which they called patch clamp [33,34]. Applying the latter to a frog skeletal muscle, they resolved for the first time single channel currents flowing across the membrane. For their invention, Sakmann and Neher received the Nobel Prize in Physiology and Medicine in 1991.
In the patch clamp technique [2,35], a tip of a pipette is pressed onto the cell surface, isolating a small patch of the membrane. Then, suction is applied, establishing a high resistance seal. The latter allows ionic current to flow only into the pipette. As the seal resistance increases with decreased surface area, electrical isolation of the small patch reduces leakage sufficiently so that even small ionic currents flowing through a single ion channel could be detected. Several configurations of patch clamp are known [2,35] (see Figure cell-attached, whole-cell, inside-out and outside-out. In the cell-attached patch clamp (see Figure, the pipette is sealed to the membrane patch, leaving the cell intact. Ionic current of channels residing within the patch is monitored. The solution inside the pipette is controlled, which makes this configuration appropriate for investigating ion channels’ modulation by various compounds. In the whole-cell patch clamp (see Figure, a pulse of suction or voltage ruptures the patch. Hence, in contrast to the cell-attached patch clamp, ionic current of the entire membrane is monitored. Additionally, the membrane voltage can be controlled via the voltage clamp. Withdrawing the pipette from the rest of the cell results in a cell-excised configuration (see Figure, D), allowing one to control both the intracellular and extracellular environments of the patch. Pulling the pipette from the cell-attached or whole-cell configurations establishes the inside-out or outside-out configurations, respectively. In the inside-out patch clamp (see Figure, the cytosolic side of the patch is exposed to the bath solution. This configuration is applied for studying ion channels regulated by intracellular ligands. In the outside-out patch clamp (see Figure, outside of the membrane faces the bath solution. This configuration is used to study ion channels regulated by extracellular ligands.

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The first crystal structure of an ion channel

Though experimental methods such as site-directed mutagenesis provided an extremely valuable insight into the architecture of ion channels, the latter remained rather obscure until the end of the XX century, when the first crystal structure of an ion channel was resolved. MacKinnon and colleagues succeeded to crystallize the transmembrane domain of a bacterial pH-dependent potassium channel KcsA from Streptomyces lividans and to resolve its atomistic structure [48,49]. For this breakthrough, MacKinnon was further awarded the Nobel Prize in Chemistry in 2003. Revealing the atomistic structure of KcsA initiated a new revolution in the general understanding of structure and functioning of ion channels and, additionally, brought more scientists using molecular modeling methods such as molecular dynamics (MD) simulations into the ion channels’ field.
The structure of KcsA closely resembles the pore domain of mammalian voltage-gated potassium (Kv) channels [50] and, therefore, may be a good model for studying their mechanisms of gating and selectivity ([51– 54], rev. [55,56]). KcsA is a tetramer; each subunit has 2 α-helical TM segments called M1 and M2 (see Figure The M1 helices gathered from the four subunits encompass the conductive pore. There is also a small pore helix (P-helix) shifted to the extracellular site of the channel and following this helix loop, forming the selectivity filter [48,49].

Table of contents :

2.1. Short history of ion channel’s discovery: from the experiments of Galvani to the first crystal structure
2.1.1. Classification of ion channels
2.1.2. Toward the model of Hodgkin and Huxley
2.1.3. The Hodgkin and Huxley model
2.1.4. Overall architecture of an ion channel captured from the voltage clamp experiments
2.1.5. Patch clamp, an advanced technique for measuring ionic currents
2.1.6. Accessing the primary structure of ion channels
2.1.7. The first crystal structure of an ion channel
2.2. Voltage-gated potassium (Kv) channels
2.2.1. Action potentials from a neuron and from a cardiomyocyte
2.2.2. Typical structure of a voltage-gated potassium channel
2.2.3. Working cycle of Kv1.2, a typical voltage-gated potassium channel: the voltage sensor transitions
2.2.4. Working cycle of Kv1.2, a typical voltage-gated potassium channel: coupling to the pore domain
2.2.5. Molecular basis for coupling between the voltage sensor and the pore
2.2.6. Particular features of Kv7.1: sequence similarities and dissimilarities with Kv1.2
2.2.7. Particular features of Kv7.1: coupling
2.3. Regulation of ion channels’ functioning by the cell membrane
2.3.1. Stabilization of poorly populated ion channels’ states by the modified bilayer content
2.3.2. Non-specific regulation of ion channels’ functioning by the cell membrane
2.3.3. Lipid content may contribute to the membrane potential
2.3.4. Specific regulation of ion channels’ functioning by the cell membrane
2.3.5. PIP2 modulates functioning of many ion channels via direct interactions with their positive residues
2.3.6. PIP2 modulated functioning of the Shaker and Kv1.2 channels
2.3.7. PIP2 modulates functioning of the Kv7 subfamily
APPENDIX to Chapter 2
A2.1. Experimentally recorded curves: I/V, G/V, Q/V and F/V
A2.1.1. The ionic current and the conductance
A2.1.2. The gating current and the gating charge
A2.1.3. Tracking the voltage sensor movement by measuring the fluorescence signal of the label
A2.2. Localization of voltage-gated potassium (Kv) channels
3.1. Molecular Dynamics (MD) Simulations
3.1.1. The principle of MD
3.1.2. Ensembles of statistical thermodynamics
3.1.3. Ergodic hypothesis
3.1.4. Integration schemes
3.1.5. Force fields
3.1.6. Periodic boundary conditions
3.1.7. Calculation of non-bonded atomic interactions (Coulomb and vdW)
3.1.8. Thermostats
3.1.9. Barostats
3.2. Estimation of the free energy
3.2.1. Free energy
3.2.2. Collective variables (CVs)
3.2.3. Metadynamics
3.2.4. Well-tempered metadynamics
3.2.5. Probability distribution for unbiased CVs
3.3. Homology modeling
3.3.1. Identification of the homologues with known structures
3.3.2. Accuracy of the homology modeling from similarity between a template and a model
3.3.3. Building a model
3.3.4. Model assessment
4.1. Modulation of the Kv1.2 channel by PIP2
4.1.1. Methods: preparation of the systems for an MD run
4.1.2. Results: in Kv1.2, there are three potential sites of PIP2 binding
4.1.3. Discussion: state-dependent interaction between Kv1.2 and PIP2 rationalizes the dual effect observed experimentally
4.2. Alteration of the Kv1.2 activation and deactivation free energies induced by the presence of PIP2
4.2.1. Methods: preparation of the systems for a metadynamics run
4.2.2. Methods: devising the effective collective variable CV! »
4.2.3. Methods: re-estimation of the free energy in terms of the gating charge Q
4.2.4. Methods: protocols and parameters of a metadynamics run .
4.2.5. Results: PIP2 changes the relative stability of the voltage sensor
states and also affects the free energy barriers separating them
4.2.6. Results: characterization of the PIP2 binding to the Kv1.2 voltage sensor
4.2.7. Discussion: the Q/V curve of Shaker is right-shifted in the presence of PIP2 due to the drastic destabilization of the Υ state
4.3. Modulation of the Kv7.1 channel by PIP2
4.3.1. Methods: preparation of the systems for an MD run
4.3.2. Results: PIP2 interacts with the VSD and the PD of Kv7.1 in a state-dependent manner
4.3.3. Results: mutations of K183 and R249, the gain-of-function residues, favor the activated/open mode of protein-lipid interactions
4.3.4. Results: the S4-S5/S6 interactions are destabilized by repulsion between their positively charged residues
4.3.5. Discussion: PIP2 is prominent for Kv7.1 due to weakened interactions between S4-S5 and S6
5.1. Modulation of the kinetic constant of the Kv1.2 activation/deactivation processes by PIP2
5.2. Identification of a PIP2 putative binding site of the Kv7.1/KCNE1 complex
5.3. Building the model of the voltage-dependent phosphatase CiVSP (including the TM and C-terminal cytoplasmic domains)


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