Doctor of Philosophy

dissertationSubcellularly resolved, excitable changes (i.e., those induced by electrical or chemical stimuli) in membrane capacitance, influenced by factors including integralmembrane protein activity, lipid densities and membrane-bound water content, may be used to elucidate nonconductive ion-channel conformational state changes, lipid-raft locations and drug-membrane binding processes. However, membrane capacitance has proven difficult to measure, partially because of bandwidth limitations associated with glass/quartz pipettes used during conventional electrophysiology. To address these challenges, techniques introduced in this thesis integrate the principles of extracellular radio frequency (RF) recording with conventional two-electrode voltage clamp (TEVC) to 1) spatially resolve effective membrane capacitance and 2) monitor excitable changes in effective membrane capacitance. Furthermore, this thesis also introduces a new multielectrode method to approximate electrode-electrolyte interfacial impedance, which might prove useful in electric impedance spectroscopic or electric impedance tomographic applications. Specific contributions include the following: 1) A method that simultaneously estimates double-layer and interelectrode (chamber) impedances, in the linear regime of electrode voltage-current sensitivity, during extracellular electrode-based measurements. This method estimates impedance parameters by applying a nonlinear least-squares regression to measurements between various groups or pairs of a three-electrode system and, unlike previous double-layer approximation methods, can be done without the use of multiple calibration solutions or moveable electrode configurations. 2) A platform capable of visualizing the spatial distribution of membrane capacitance, using extracellular RF electrode recordings, around a single cell. The proof-of-concept for this technique is demonstrated with dielectric maps around polarized Xenopus oocyte membranes. 3) Development and characterization of a platform to enable RF impedancebased measurements around voltage-clamped ShakerB-IR-expressing Xenopus oocytes. Data indicated that the platform was most sensitive to effective changes in oocyte dielectric at 300 kHz and 500 kHz. 4) Temporal characterization of changes in voltage-sensitive RF membrane capacitance associated with ShakerB-IR activation (expressed in Xenopus oocytes) and ShakerB-IR-Cu2+ interactions. Results indicate that extracellular RF-impedance-based measurements can temporally and spatially elucidate changes in excitable cell-membrane capacitance and could supplement conventional electrophysiological techniques to provide a broader understanding of cellular biophysics

Monitoring Voltage-Dependent Charge Displacement of Shaker B-IR K+ Ion Channels Using Radio Frequency Interrogation

Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz) electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K+ ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents. Simultaneously, RF electric fields were applied to perturb the membrane potential about the TEVC level and to measure voltage-dependent RF displacement currents. ShB-IR expressing oocytes showed significantly larger changes in RF displacement currents upon membrane depolarization than control oocytes. Voltage-dependent changes in RF displacement currents further increased in ShB-IR expressing oocytes after ∼120 µM Cu2+ addition to the external bath. Cu2+ is known to bind to the ShB-IR ion channel and inhibit Shaker K+ conductance, indicating that changes in the RF displacement current reported here were associated with RF vibration of the Cu2+-linked mobile domain of the ShB-IR protein. Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains — capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug–protein interactions

Temporally Resolved RF Measurements.

<p>A) RF impedance changes (|ΔZ_RF|) measured during TEVC relative to the impedance at holding potential (−90 mV) in control oocytes, expressing endogenous proteins only (<i>n</i> = 10, left column), and <i>Sh</i>B-IR expressing oocytes (<i>n</i> = 9, right column). <i>Sh</i>B-IR expressing oocytes elicited a membrane-potential-dependent (V_m^*) RF response different than control oocytes. RF impedance changes were analyzed in two regions; the RF response during the onset of voltage-step (<b>o</b>, average |ΔZ_RF|_o 0–1 ms after voltage step, <i>dV_m^*/dt</i> > 0) and the RF response after membrane potential achieved its command (steady-state) level (<b>s</b>, average |ΔZ_RF|_s 5–35 ms after voltage step, <i>dV_m^*/dt</i> ≅ 0). B) TEVC current measurements were used to verify ion-channel expression and responses (leak current subtracted, capacitive transient unsubtracted) to C) whole-cell voltage-clamp.</p

Set-up and circuit model.

<p>A) Changes in RF membrane impedance (|ΔZ_RF|) during TEVC were measured by passing RF current from an electrode surrounding the meridian of the cell (black) to a ground electrode (media, above the cell). Contour lines and colors of the saggital cross-section of a cell in the recording chamber, shown here, illustrate the general spatial distribution of the RF electric potential expected based on the Maxwell equations for a passive cell under axisymmetric conditions (π/4 phase shown). B) A circuit model of the chamber including the shunt resistance (R_s,), membrane impedance (Z_m), intracellular resistance (R_i), and electrode double layer (Z_dl). C) Using the circuit model, the frequency-dependent RF impedance would change (Δ|Z_RF|) with an increase or decrease in membrane capacitance — a change that would be most easily detectable at frequency ω* where the maxima of the |ΔZ_RF| occurs. The present study reports changes in RF impedance |ΔZ_RF| evoked by TEVC step changes in membrane potential.</p

Steady-state RF response for <i>Sh</i>B-IR and Control Cells.

<p>A) Significant (^x<i>p</i> = .1, *<i>p</i> = .05) voltage-dependent differences in |ΔZ_RF|_s were observed between control oocytes, expressing endogenous proteins only (“Endo”, orange), and <i>Sh</i>B-IR expressing oocytes (express both endogenous and <i>Sh</i>B-IR proteins, “<i>Sh</i>B-IR + Endo”, brown). The “Endo” response was subtracted from the “<i>Sh</i>B-IR + Endo” response to estimate the isolated RF response from the <i>Sh</i>B-IR channels only (“<i>Sh</i>B-IR only”, blue). Error bars denote +/− standard errors of the mean (SEM). B) The SEM for the isolated <i>Sh</i>B-IR proteins (“<i>Sh</i>B-IR only”, blue) was also estimated by subtracting the SEM from the control oocytes (“Endo”, orange) from the SEM associated with the <i>Sh</i>B-IR expressing oocytes (“<i>Sh</i>B-IR + Endo”, brown). The SEM for isolated <i>Sh</i>B-IR expressing oocytes was largest near the half-activation potential for these ion channels. C) <i>Sh</i>B-IR channel expression and voltage-dependent whole-cell current was verified using TEVC, and this data was used to estimate <i>Sh</i>B-IR conductance (G/G_max, inset).</p

Onset RF Response in <i>Sh</i>B-IR Expressing Oocytes.

<p>Changes in RF impedance during the onset of voltage-clamp (|ΔZ_RF|_o, 0–1 ms after whole-cell depolarization) were slightly depressed in control oocytes with the addition of Cu²⁺ (Cu²⁺-free - green markers, Cu²⁺ addition - purple markers), but were significantly greater in <i>Sh</i>B-IR expressing oocytes (Cu²⁺-free - green line, Cu²⁺ addition - purple line).</p

Copper treatment and steady-state <i>Sh</i>B-IR RF response.

<p>A) Voltage-dependent differences in |ΔZ_RF|_s were observed between <i>Shaker</i> expressing oocytes (“<i>Sh</i>B-IR + Endo”, green line) and the same cells exposed to ∼120 µM Cu²⁺ (purple line). A similar effect was apparent, albeit to a lesser extent, for control cells before (“Endo”, green markers)/after Cu²⁺ treatment (purple markers). Error bars denote +/− standard errors of the mean (SEM). B) Even though RF charge displacements increased in Cu²⁺-exposed <i>Sh</i>B-IR expressing oocytes, TEVC whole-cell current decreased showing that Cu²⁺ successfully blocked the channels (channel conductance shown as inset).</p