Ponce A et al. (2013), Voltage-dependent potassium currents expressed ...

XB-ART-48909

Physiol Rep 2013 Dec 08;17:e00186. doi: 10.1002/phy2.186.

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Voltage-dependent potassium currents expressed in Xenopus laevis oocytes after injection of mRNA isolated from trophozoites of Giardia lamblia (strain Portland-1).

Ponce A , Jimenez-Cardoso E , Eligio-Garcia L .

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Despite its importance as a health problem issue, almost nothing is known about the membrane physiology of Giardia lamblia and practically there exist no information so far regarding the variety and properties of ion channels that this protozoan parasite possesses. To address this subject we resorted to an indirect method, consisting in the injection of mRNA and further characterization of ion currents in Xenopus oocytes. In this work, we show that oocytes injected with mRNA isolated from cultured trophozoites of G. lamblia, strain Portland-1 express novel potassium currents that appear over the second day after injection and show time- and voltage-dependent activation followed by a slow inactivation. They start activating at -90 mV, with V1/2 of -30 mV; its time constant of activation (at +60 mV) is 0.11 sec, whereas that of inactivation is 1.92 sec, V1/2 = -44.6 mV. Such K currents were effectively blocked by K channel blockers TEA and 4AP, as well as Ba(2+), quinine, quinidine, charybdotoxin, dendrotoxin-1, capsaicin, margatoxin, and diltiazem. These results suggest that such currents are the result of expression of Giardia's voltage-gated K channels heterologously expressed in Xenopus laevis oocytes.

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Species referenced: Xenopus laevis

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	Figure 1. Effect of Giardia lamblia mRNA injection on the membrane potential (Em) of Xenopus oocytes. Bar length indicates the mean value (±SE) of membrane potential of individual mature oocytes that were either noninjected or injected with water, degraded mRNA or intact mRNA. Negative values are shown upward for convenience. Em measurements were made on oocytes 3 days after injection. **P < 0.005 for a t‐test, comparing noninjected versus injected with mRNA. Numbers above bars indicate number of cases. Multiple comparisons test (Tukey) showed no statistically significant difference among noninjected values with neither injected with water nor injected with degraded mRNA.
	Figure 2. Expression of exogenous ion currents by injection of oocytes with mRNA isolated from Giardia lamblia. (A) Representative series of traces of current obtained from oocytes under the distinct experimental conditions indicated on the legends, they all are depicted at the same magnitude indicated in the scale bar at the top. Currents were obtained in response to series of squared voltage pulses that as shown in (B) were from −120 up to +80 mV in steps of 20 mV from a holding level of −80 mV. (C) Relationship between the maximal current amplitude and the test voltage from oocytes injected with intact mRNA as well as noninjected or mock injected. Dots indicate the average value (±SE) of the number of individual oocytes assayed that the inset indicates. (D) Time course of the expression of currents after days of injection. Bar length indicates the mean value of peak current in response to a voltage pulse of +60 mV.
	Figure 3. External K+ dependence of reversal potential. (A) Series of tail currents, at distinct external K+ concentrations, obtained from an oocyte injected with mRNA, in response to a stimulus protocol that, as depicted in (B), after a conditioning pulse of +60 mV, varies test voltage from −120 up to 10 mV in steps of +10 mV. (C) The relationship between the tail current amplitude and the test voltage shows a linear trend and shifts right when potassium is increased on the external media, as expected for a K+ selective pathway. (D) Semi‐log10 plot of the calculated reversal potential versus the external K+ concentration (empty circles) follows a linear trend with a slope of 55 mV per tenfold increase of K+, a value nonsignificantly distinct from the ideal value of 59 mV per tenfold (filled circles).
	Figure 4. Kinetic properties of mRNA‐induced K+ currents. (A), (C), and (E) show in the upper part representative series of currents obtained from oocytes injected with Giardia lamblia mRNA in response to the voltage stimulus protocols indicated in the lower part. (A) In order to determine the voltage dependence of its activation, the instant magnitude of the tail current was measured at each test potential. (B) The standardized value of such current was averaged (25 oocytes from 3 frogs) and plotted against the test potential. (C) To determine the voltage dependence of the steady‐state inactivation, the peak value of current was measured at +60 mV after prepulses of variable voltage. (D) The mean value (23 oocytes from 3 frogs) of the standardized peak current plotted against the test voltage produces a sigmoid relationship. (E) Representative current traces from a protocol designed to determine the time recovery of inactivation. (F) Time‐dependent recovery of inactivation. To obtain this plot, the peak magnitude of the current of the second pulse was divided by the peak magnitude of the current of the first pulse and the average value of this ratio (from 18 oocytes) was plotted against the interpulse time lapse. This relationship follows an asymptotically exponential rise with a time constant of 40.5 sec.
	Figure 5. Effect of TEA and 4AP on Giardia lamblia′s‐mRNA‐induced K+ currents. (A) Representative traces showing the effect of K channel blockers TEA (top) and 4‐AP (bottom). Ion currents were elicited by a pulse of +60 mV (for 10 sec, from a holding of −80 mV) while oocytes were in a media containing drugs in increasing concentrations. From such recordings a %blocking index was calculated by referring the peak current at each drug concentration to the peak current without drugs. (B) In both cases the averaged% blocking (15 oocytes from 3 frogs each) shows a sigmoidal trend when values are plotted against the log10 of drug concentration.

References [+] :

Adam, The biology of Giardia spp. 1991, Pubmed