Patch-clamp recordings and EPR measurements

For electrophysiologists, patch-clamp recordings are the method of choice for studying ion channels (Neher & Sakmann, 1976). Membrane patches are analyzed concerning their response to different membrane voltages induced by changes in the ion composition of the pipette solution or the patch external environment. In detail, the membrane potential Vm

and equilibrium potential Veq for a given ion are unequal in a real cell. Consequently, an electrochemical driving force VDF pushes each contributing ion into its equilibrium state (Equation 2).

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𝑉_𝐷𝐹 = 𝑉_𝑚− 𝑉_𝑒𝑞 Equation 2

The arithmetic sign of the driving force in combination with the valence of the ion under investigation is used to define the direction of ion movement across the membrane.

Additionally, the driving force can be used for measuring currents Im of specific voltage-sensing membrane proteins, for calculating the ion conductance G, known as a function of the total number of open channels for a specific ion, and for determining the reversal potential Vrev of an ion channel (equal to the resting potential Vrest of a cell) (Figure 6).

Currents can be measured and translated to single channel activities. To this end, measured currents are plotted against the different membrane potentials (Figure 6). Currents were normalized (I/Imax) and fitted by a two-state Boltzmann equation to determine the voltage at which half of the channels are open (Vmid) to estimate their opening probability. Patch-clamp experiments enable a direct analysis of measurable currents and flow directions.

HV1 channels were extensively studied using the patch-clamp technology. To this end, channels were overexpressed in different cell types (COS/HEK cells, xenopus leavis oocytes) and analyzed concerning their gating behavior (Ramsey et al., 2010; Musset et al., 2011;

Morgan et al., 2013; Hong et al., 2014a; Mony et al., 2015).

Figure 6: Hypothetical current-voltage (I-V) relationship for studying voltage-sensing membrane proteins.

The plot represents measured currents Iion in a voltage-clamp recording under changes in the membrane potential Vm (-160 to 80 mV). To this end, the intersection with the X-axis is defined as the reversal potential V_rev. If the measured currents are caused by one defined ionic species the reversal or resting potential corresponds to the Veq of this ion (Iion = Gion (Vm-Veq)). Consequently, the ionic species channeled/transported can be analyzed and the direction of ion flow can be determined. For values smaller than Veq, the ion enters the cell whereas at more positive values than V_eq the ion flows out.

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I-V-curves of hundreds of mutants were recorded and evaluated (Ramsey et al., 2006; Sasaki et al., 2006; Tombola et al., 2008; Musset et al., 2010a; Ramsey et al., 2010; Tombola et al., 2010; Gonzalez et al., 2010; Musset et al., 2011; Smith et al., 2011; Berger & Isacoff, 2011;

Hong et al., 2013; Morgan et al., 2013; Hondares et al., 2014; Hong et al., 2014a; Fujiwara et al., 2014; Cherny et al., 2015; Chamberlin et al., 2015; Mony et al., 2015; Chaves et al., 2016;

Okuda et al., 2016; DeCoursey et al., 2016). For example, experiments with hHV1 revealed a change in cation to anion selectivity when D112 was exchanged to a neutral charged aa (1.2.1) (Musset et al., 2011; Morgan et al., 2013). Mutations of aspartate to glutamate retain cation selectivity (DeCoursey et al., 2016). Further electrophysiology studies highlighted different countercharge positions in the transmembrane residues in the closed or open state of the channel (DeCoursey et al., 2016). Nevertheless, the patch-clamp technique cannot be used exclusively to determine mechanistic details. For example, the complexation of polyvalent cations by two His residues in hHV1 is controversially discussed (1.2.3). In patch-clamp recordings, it was shown that, a mutation of His to Ala prevented Zn²⁺ binding (Cherny & DeCoursey, 1999; Ramsey et al., 2006). Nevertheless, later, homology models revealed a much more complex binding event whereby the dimer interface in combination with the His residues acts as the anchor point for zinc binding (Musset et al., 2010c).

Voltage-clamp fluorometry as a further development of the patch-clamp technology, enables the simultaneous preservation of structural and functional data of the protein under investigation (Kalstrup & Blunck, 2017; Wulf & Pless, 2018), making it an ideal tool for studying HV1 channels. The HV1 channel from ciona intestinalis (CiHV1) labeled with Alexa-488 maleimide revealed cooperative gating in dimeric channels. Furthermore, detailed analysis of the Zn²⁺ inhibition properties supposed the existence of two Zn²⁺ coordination sites in a monomeric channel (Qiu et al., 2013; Qiu et al., 2016). The Zn²⁺ binding blocks the channeling of protons in one site and hinders the movement of S4 in another (Qiu et al., 2016).

Information about the tertiary or even quaternary protein structure can be gained by using the patch-clamp technique in combination with paramagnetic agents. To this end, accessibility studies have been performed with single-cysteine mutants of HV1 overexpressed in cells. Here, the rate of e.g. methanethiosulfonate (MTS) binding to cysteine residues is proportional to their accessibility. The results suggested a parallel movement of S1 and S4 (Gonzalez et al., 2010; Mony et al., 2015).

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Additionally, paramagnetic agents can be used to study oligomeric states in combination with amino acid exchanges by applying the electron paramagnetic resonance (EPR) spectroscopy technique. Here, electron spins of radicals are exposed to microwaves by concurrent increase of the magnetic field strength and measuring their resonance absorption. To this end, the motion of a spin label attached to a cysteine residue of a single-cysteine mutant (protein) can be calculated, known as the mobility (ΔHo−1

). A high mobility is a hint for loop regions. Additional paramagnets like e.g. nickel ethylenediamine-N,N'-diacetic acid (NiEDDA) and oxygen are used for accessibility (Π) screenings. In this case, high ΠO2 values indicate lipid contacts, high ΠNiEDDA values, indicate contact to solvent outside the membrane, mostly loop regions (low ΠNiEDDA indicate the transmembrane region). Analyzing such data for single-cysteine mutants of hHV1 reconstituted in liposomes revealed the non-lipid contacts of D112, R205, R208 and R211, the individual length of each helix in the given membrane environment and the presence of water-filled crevices (Li et al., 2015).

Nevertheless, movements can occur in any direction of the x-, y- and z-axis, whereby the accessibility will always be changed, which would hamper a precise interpretation.

Therefore, often the EPR-obtained information are combined with MD simulations.