Electrophysiology

Chapter 1: General introduction

1.4 Tools for systematic analysis of ion channel function

1.4.2 Electrophysiology

To determine the effects of both canonical and non-canonical amino acid mutagenesis on receptor function, we used two-electrode voltage clamp (TEVC). In TEVC, the cell is impaled with two microelectrodes: one measures the membrane potential, while the other injects current to maintain the membrane potential at a constant, preset potential (Figure 1.11A).¹⁰⁶ Upon agonist application to the cell, ion channels will open and the membrane potential changes as a result of the ion flow in or out of the cell. The current injected through the current electrode is therefore a direct measure of the sum of currents through all ion channels at the membrane. Electrophysiology assays require only a small amount of protein for signal responses (microAmpères), which makes it an extremely useful tool to measure influences of subtle perturbations on receptor function.¹⁰⁷

A commonly used heterologous expression system to study ion channels is the oocyte from Xenopus laevis (African clawed frog). This vertebrate cell is a classic expression system for functional evaluation of ion channels using TEVC, because of its size and robust expression of eukaryotic proteins.¹⁰⁸ Proteins are expressed by microinjecting the mRNA coding for the desired protein into the cell, followed by an incubation period of 24-48 hours, during which the (mutant) protein is translated, assembled and transported to the cell membrane.^104,109 The microinjection delivery method makes this approach quite amenable for the nonsense

suppression approach described earlier. For in vivo nonsense suppression, both mRNA and the chemically acylated tRNA are readily co-injected in Xenopus laevis oocytes (Figure 1.10).

Figure 1.10 | In vivo nonsense suppression using a Xenopus laevis oocyte.

Different aspects of ion channel function may be evaluated using various electrophysiological assays. For all studies in this dissertation regarding pLGICs, the main goals were to assess functional effects of mutants and determine potency changes among substituted ligands. For activation of pLGICs the following events are considered: the cooperative binding of one or more agonists (binding) allows the receptor to undergo conformational changes towards an ion-permeable state (gating). The introduction of mutants or application of ligands may influence the energetics of agonist binding or gating, which results in a change in sensitivity of the pLGIC to its agonist.¹¹⁰ Generally, researchers assume that mutations near the agonist binding site influence only the binding event, while those close to the pore are thought to mostly influence the gating event.⁴⁵ To measure these effects, we

performed dose-response experiments of which a typical protocol is shown in Figure 1.11B.

Upon application of increasing agonist doses, the binding equilibrium is shifted towards the open state. At high doses, saturation is reached and current responses level off. Peak currents are normalized to the maximal response and plotted against the logarithm of the respective agonist concentrations. We then fit these data to the Hill equation:

𝐼([𝐴]) = 𝐼_𝑚𝑎𝑥 1 + (𝐸𝐶[𝐴] )^{50 𝑛𝐻}

Where I is the whole cell current, [A] is the agonist concentration, Imax is the maximal current response, EC50 is the agonist concentration eliciting a half-maximal response, and nH is the Hill coefficient (Figure 1.11C). The shift in EC50 compared to wild type is used as a readout for the magnitude of the energetic perturbation caused by the mutations or ligand substitution, where log(fold shift in EC50) varies roughly with the ΔΔG value.

A different electrophysiology assay that renders information on receptor properties is a voltage jump experiment. For nAChRs described in chapter two, this assay is used to verify receptor stoichiometry. The two α4β2 stoichiometries demonstrate distinct behaviors at positive membrane potentials.⁴⁵ Upon nAChR activation, the negative membrane potential drives ion flow into the cell. Ion channel opening at positive potentials results in an outward current of ions. However, the α4β2 A2B3 stoichiometry prevents this outward flow at positive potentials, which we refer to as inward rectification (Figure 1.11D). Since we control the membrane potential in TEVC, we can change the membrane potential quickly to positive potentials upon receptor activation to measure the extent of inward rectification (Figure 1.11E). This allows us to distinguish between α4β2 stoichiometries.

Figure 1.11 | Functional characterization of ion channels using two-electrode voltage clamp (TEVC). (A) Schematic presentation of the TEVC set up using a Xenopus laevis oocyte. (B) Dose- response protocol: current waveforms in response to agonist. Application of increasing doses of agonists results in increasing current responses. (C) Dose-response relationships illustrating wild type (black), gain of function (blue), loss of function (red) variants. (D) I-V plot of a voltage jump experiment on a pLGIC. At negative potentials, the I-V relationship is linear (blue). At positive potentials, this trend can either continue (green) or be inhibited (red). This phenomenon of current inhibition at positive potentials is called inward rectification. (E) Voltage jump protocol. Although this protocol differs slightly when used for pLGIC or VGICs, the concept is the same: the holding potential of the membrane is varied on a millisecond timescale and the currents for each voltage step are recorded. For voltage jump experiments on pLGICs, the voltage jump is executed upon agonist application. (F) G-V plot resulting from a voltage jump experiment on VGSCs. Sodium conductances were calculated from peak currents, membrane potential and the reversal potential.

Besides pLGIC characterization, we also used voltage jump experiments to determine activation characteristics of VGICs in chapter four. As depolarization of the membrane activates VGICs, a typical experiment involves varying the membrane potential in 5 mV

depolarization steps to obtain a current-voltage plot for VGKCs and a conductance-voltage plot for VGSCs. We determine sodium conductances (GNa) from peak currents using the equation:

𝐺_𝑁𝑎(𝑉_𝑚) = 𝐼_𝑁𝑎 (𝑉_𝑚− 𝑉_𝑟𝑒𝑣)

where GNa is the sodium conductance, INa is the peak current at a that depolarization step, Vm

is the membrane potential, and Vrev is the reversal potential calculated for each individual cell.

The reversal potential is the membrane potential at which the driving force for an inward sodium current – i.e. the concentration gradient of sodium ions – is equal to the electrical potential. We normalized individual conductance curves (G-V) to the maximum conductance amplitude per cell and fitted using the Boltzmann distribution equation:

𝐺(𝑉_𝑚) = 𝐺_𝑚𝑎𝑥 1 + 𝑒^{𝑉−𝑉𝑘𝑜.5}

where G is the normalized sodium conductance, Vm is the membrane potential of that depolarization step, V0.5 is the half-activation potential, and k is the slope. V0.5 represents the membrane potential at which half of the channel population is activated, which we used as a measure for the voltage-dependence of activation (Figure 1.11F).