Corbin-Leftwich A et al. (2018), A Xenopus oocyte model system to study action p...

XB-ART-55333

J Gen Physiol 2018 Nov 05;15011:1583-1593. doi: 10.1085/jgp.201812146.

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A Xenopus oocyte model system to study action potentials.

Corbin-Leftwich A , Small HE , Robinson HH , Villalba-Galea CA , Boland LM .

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Action potentials (APs) are the functional units of fast electrical signaling in excitable cells. The upstroke and downstroke of an AP is generated by the competing and asynchronous action of Na+- and K+-selective voltage-gated conductances. Although a mixture of voltage-gated channels has been long recognized to contribute to the generation and temporal characteristics of the AP, understanding how each of these proteins function and are regulated during electrical signaling remains the subject of intense research. AP properties vary among different cellular types because of the expression diversity, subcellular location, and modulation of ion channels. These complexities, in addition to the functional coupling of these proteins by membrane potential, make it challenging to understand the roles of different channels in initiating and "temporally shaping" the AP. Here, to address this problem, we focus our efforts on finding conditions that allow reliable AP recordings from Xenopus laevis oocytes coexpressing Na+ and K+ channels. As a proof of principle, we show how the expression of a variety of K+ channel subtypes can modulate excitability in this minimal model system. This approach raises the prospect of studies on the modulation of APs by pharmacological or biological means with a controlled background of Na+ and K+ channel expression.

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Species referenced: Xenopus laevis
Genes referenced: dtl nav1 sh2b2

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	Figure 1. A reliable approach to recording APs in Xenopus oocytes. (A and B) Schematic of the loose-clamp electronics used to measure APs with a TEVC amplifier. The dashed red box shows the adapted circuitry, which is enlarged in B. Electrical recordings in oocytes injected 2â4 d prior with an RNA mixture for sodium and potassium channels: 10 ng Nav1.4, 2.5 ng NavÎ²1, 5 ng Kv7.2, 5 ng Kv7.3, and 0.6 ng ShakerÎ. (C) In the loose clamp, 1-ms voltage pulses were applied to depolarize the membrane. Top: Membrane current produced by the depolarizing stimulus. Bottom: Membrane depolarization. The inset in C shows a subthreshold depolarization (black trace), and the next pulse stimulated a voltage change that is an all-or-none AP (red trace). (D) For the same RNA mixture as in C, the APs in oocytes were examined with paired pulses to measure the refractory period. The 1-ms pulses (top) were 16 ms apart for the traces on the left. The black traces for the pulses were for stimuli of the same amplitude, whereas red traces signify that the second pulse was of greater amplitude than the first. The corresponding membrane voltage recordings are shown in the bottom left with the same color coding. On the right side, the 1-ms depolarizing pulses (top) were 9 ms apart. The black traces were for stimuli of the same amplitude, whereas blue traces signify that the second pulse was of greater amplitude. The corresponding membrane voltage recordings are shown in the bottom right. The second stimulus was increased four times without eliciting an AP.
	Figure 2. Testing for AP generation in oocytes after expression of sodium channel RNA only. Oocytes were injected with 10 ng Nav1.4 and 2.5 ng NavÎ²1 subunits only and recorded 2â4 d postinjection. K+ channel RNA was not included. (AâD) Representative membrane potentials recorded from oocytes maintained at a membrane voltage of approximately â50 mV, in response to stepwise increases in depolarizing pulses (1 ms) in the loose clamp recording mode. The insets in each panel show TEVC current recordings (not leak-subtracted) of the estimated maximum inward Na+ current recorded for each oocyte: 1 ÂµA (A), 17 ÂµA (B), 25 ÂµA (C), and 43 ÂµA (D). Voltage and current recordings in AâD are all on the same axis scales, for comparison of amplitudes and kinetics. The upward current deflections in the insets (AâD) are unsubtracted capacitive artifacts (truncated in BâD). These are representative examples from among a larger population of oocytes showing variable levels of Na+ current expression. (E and F) The probability of firing an all-or-none AP was determined for all cells injected with Na+ channel Î± and Î² RNAs only and expressing <20 ÂµA (E) or â¥20 ÂµA (F) of peak inward current. AP genesis was determined in response to variable-amplitude 1-ms depolarizing pulses in the loose-clamp mode for VREST values between â42.5 and â72.5 mV (grouped in increments of 5 mV). The number on each bar represents the number of recordings at each range of VREST. The dashed lines in E and F designate the maximum probability of firing an AP for the conditions of low (E) and high (F) Na+ current expression.
	Figure 3. TREK (K2P) channel expression affects AP duration and VREST. (A) APs recorded in oocytes after expression of Nav channels (Nav1.4 Î±, 10 ng; NavÎ²1, 2.5 ng) plus 5 ng each of Kv7.2 and Kv7.3 or Nav channels plus TREK-1 (right, 3.0â6.5 ng E306A mutant; see Methods). The insets in A show the Kv7 or TREK-modified AP on different time scales. 3 d after RNA injection and incubation, the membrane potential recordings were done from a VREST of â60 mV using 1-ms depolarizing pulses. (B) APs from four different oocytes with low (blue trace) to high (orange trace) expression of TREK currents are shown. Inset: The half-width of each AP was measured and plotted as a function of the estimated magnitude of the outward TREK current at +35 mV. In the inset, the four symbols in color (orange, green, pink, and blue) correspond to the traces shown on the left for low to high TREK currents, respectively. (C and D) VREST was recorded after penetration of both recording pipettes into oocytes expressing Nav channels with the addition of the Kv7.2/7.3 subunits (red) or TREK (blue). Box plots show 25â75% of the range (the box); the solid square inside the box is the mean VREST, and the whiskers depict the maximum and minimum values. The line within the box represents the median value. Complete data for VREST measured in cells expressing Nav channels plus TREK (blue box plot) are expanded in D for individual cells with outward currents ranging from 3 to â¼20 Î¼A at a command potential of +35 mV in TEVC.
	Figure 4. Maintenance of VREST by inwardly rectifying K+ channels regulates AP generation in Xenopus oocytes. Xenopus oocytes were injected with an RNA mixture of 10 ng Nav1.4, 2.5 ng NavÎ²1, 2.5 ng ShakerÎ, and 10 or 20 ng Kir2.1 and incubated for 3 d before experiments. (AâE) Loose-clamp AP recordings were done using a ramp to depolarize the membrane in the absence of Ba2+ (2 Ko control; A), plus 30 ÂµM Ba2+ (B), plus 100 ÂµM Ba2+ (C), and upon recovery from Ba2+ block of Kir current by washout with 2 Ko (D). The dashed, vertical line in AâD designates the approximate time of onset of the first AP in a ramp under control conditions. Arrows in AâD designate the value of VREST for each recording condition. (E) Comparison of VREST measured before the current ramp that elicited APs, recorded as in AâD. The impact of 30 and 100 ÂµM Ba2+ was measured (n = 6); significant differences from control values are noted by *. Box plots show 25â75% of the range (the box); the solid square inside the box is the mean VREST, and the whiskers depict the maximum and minimum values. The line within the box represents the median value. (F) For the same cells as in AâE, the expression of the Kir2.1 channel current was observed in TEVC recordings using a 10 Ko solution (black trace) and a voltage ramp to elicit the K+ currents. The currentâvoltage plot shows an example of block of the inward Kir current by 30 ÂµM Ba2+ (blue trace) and 100 ÂµM Ba2+ (red trace). The recovery is in 10 Ko (gray trace). The inset quantifies the percentage of the 10 Ko inward current at â70 mV that was blocked by 30 or 100 ÂµM Ba2+ (n = 6).
	Figure 5. Diversity of Kv channel expression modifies the oocyte AP waveforms. (AâF) Representative APs evoked in oocytes 3 d after injection with RNA mixtures for Nav1.4 Î± (10 ng) and NavÎ²1 (2.5 ng) plus Kv channels. The RNAs were mixed to achieve an injection of 20 ng Kv4.2Î2-40 (A); 2.5 ng Kv7.2 and 2.5 ng Kv7.3 (B); 20 ng Kv4.2Î2-40 plus 2.5 ng Kv7.2 and 2.5 ng Kv7.3 (C); 0.65 ng ShakerÎ (D); 5 ng Kv7.2 and 5 ng Kv7.3 (E); and 0.65 ng ShakerÎ plus 5 ng Kv7.2 and 5 ng Kv7.3 (F). In AâF, each set of three membrane voltage recordings shows one response which was just below threshold (blue trace), the first suprathreshold response (red trace), and a suprathreshold response for which increasingly larger pulses of 1-ms duration failed to change the membrane potential recording (black trace). AâF use the same voltage and time axes for ease of comparison; the insets in D and F show the faster APs on a different time scale.
	Figure 6. Oocyte recordings may demonstrate multiple APs. 3 d before AP recordings, oocytes were injected with a mixture of 10 ng Nav1.4 Î± and 2.5 ng Nav1.4 Î² plus coexpressing K+ channel RNAs, as noted. (A) Spontaneous APs in oocytes coexpressing ShakerÎ (0.65 ng RNA) and Kir 2.1 (10 ng RNA). This is representative of four similar recordings from oocytes injected with the same mixture of RNAs. (B) Oscillatory APs were also spontaneously evoked in oocytes coexpressing Kv7.1 (2.5 ng RNA). This is representative of five similar recordings from oocytes injected with the same mixture of RNAs. (C) High-frequency AP firing in oocytes coexpressing ShakerÎ (0.65 ng RNA injected) and Kir 2.1 (10 ng) generated by a 1-s suprathreshold depolarizing stimulus applied from a loose clamp. Representative of four cells.

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