Patch clamp, an advanced technique for measuring ionic currents

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Table of contents

1. INTRODUCTION / INTRODUCTION
2. ION CHANNELS / CANAUX IONIQUES
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. METHODS / MÉTHODES
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. RESULTS / RÉSULTATS
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. PERSPECTIVES / PERSPECTIVES
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)
REFERENCES