Ponce A et al. (2019), Injection of mRNA isolated from trophozoites of...

XB-ART-56021

Physiol Rep 2019 Jun 01;711:e14029. doi: 10.14814/phy2.14029.

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Injection of mRNA isolated from trophozoites of Giardia intestinalis induces expression of three types of chloride currents in Xenopus laevis oocytes.

Ponce A , Ogazon Del Toro A , Jimenez L , Eligio-Garcia L , Jimenez-Cardoso E .

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Giardia lamblia is one of the most important worldwide causes of intestinal infections, yet little is known about its cellular physiology, especially the diversity of ionic channels that this parasite expresses. In this work, we show that injection of mRNA isolated from trophozoites of Giardia, into Xenopus laevis oocytes, induces expression of three types of chloride currents (here referred to as ICl-G1, ICl-G2, and ICl-G3), which have different biophysical and pharmacological properties. ICl-G1 currents show inward rectification and voltage dependence are enhanced by hypotonicity, show a selectivity sequence of (I > Br > Cl > F), and are inhibited by NPPB, DIDS, SITS, 9AC, DPC, and Zinc. These findings suggest that ICl-G1 is the result of expression of chloride channels related to ClC2. ICl-G2 currents show outward rectification and are dependent of intracellular calcium, its selectivity sequence is (Cl > Br > I > F) and are inhibited by NPPB, DIDS, SITS, 9AC, DPC, niflumic acid, tannic acid, and benzbromarone. These findings suggest that they are produced by calcium dependent chloride channels (CaCC). The third type of currents (ICl-G3) appears only after a hypoosmotic challenge, and has similar properties to those described for ICl-swell, such as outward rectification, instant activation, and slow inactivation at large depolarizing voltages. They were blocked by NPPB, DIDS, 9AC, NIf, DCPIB, and tamoxifen. Our results indicate that Giardia intestinalis has at least three types of anion conductances.

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Species referenced: Xenopus laevis
Genes referenced: dtl neff1 nppb
GO keywords: chloride channel activity

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	Figure 1. Injection of mRNA, isolated from cultured trophozoites of Giardia intestinalis, into Xenopus laevis oocytes, induces expression of exogenous chloride currents. (A) Representative series of currents obtained, in response to a voltage clamp protocol, shown in the lowest part, from oocytes that were either noninjected, injected with mRNA of Giardia, or cleaved mRNA. (B) Relationship between the average amplitude of current and the testing membrane voltage of each of the three experimental conditions (C–D) Time course of expression of the outward component of current after injection of Giardia mRNA. Bars show the average (±SE) magnitude of current at +80 mV (C) or at −140 (D) obtained from noninjected oocytes (yellow), or injected with cleaved mRNA (green) or intact mRNA (red). One‐way ANOVA , run on each group, indicates significant differences for intact mRNA‐injected oocytes (P < 0.01).
	Figure 2. Hyperpolarizing conditioning voltage enhances the inwards component of current (ICl‐G1). (A). Representative series of currents obtained, from the same oocyte, either control (left column) or injected with mRNA (right column), in response a voltage clamp protocol (depicted in the lower part of left column), consisting of step pulses ranging from −140 to + 80 mV in steps of 20 mV. Each testing pulse was preceded by a prepulse of 0, −80, or −140 mV, during 5 sec followed by an interpulse of 0 mV during 0.2 sec. In the upper row a prepulse of 0 mV was given; In the middle one a prepulse of −80 mV, and in the lower it was −140 mV. (B) I–V relationship of the three conditions described, from control (left) and mRNA‐injected oocytes. The latter shows a change of conductance in the inwards part, but not in the outwards one. (C) Comparison of the average slope conductance at −140 and + 80 mV, from control (upper) and mRNA‐injected (lower) oocytes obtained from recordings with a prepulse of either −140, −80, or 0 mV. One‐way ANOVA indicates statistically significant differences for conductance of mRNA‐injected oocytes at −140 (P < 0.01) but not at +80 mV. The same statistical analysis indicates no statistically significant difference for conductances of control oocytes.
	Figure 3. The outward component of current (ICl‐G2) depends of the intracellular calcium concentration. (A) Representative series of currents from control (upper row) and mRNA‐injected (lower row) oocytes, obtained under the experimental conditions described in the upper part. (B) I–V relationship of control (left) and mRNA‐injected (right) oocytes treated with the experimental conditions described in the upper part of figure 5. (C) Comparison of the average slope conductance at −140 (blue) and +80 mV (red) of control (left) and mRNA‐injected oocytes. one‐way ANOVA within groups indicates statistically significant differences for treatments at −140 mV (P < 0.01) but not for treatments at +80 mV of both control and injected mRNA. (D) (left) Comparison of the magnitude and kinetics of currents obtained in response to a test pulse of + 80 mV of control (red) and mRNA‐injected (blue) oocytes. (middle) Currents are shown standardized to compare the kinetics of activation. (right) Statistical comparison of the average value of the time constant (tau), obtained from fitting the standardized currents at +80 mV of control (red) and mRNA‐injected currents, indicates a significant difference (P < 0.01).
	Figure 4. ICl‐G1 and ICl‐G2 have different selectivity sequences. (A) representative traces of current obtained in response to ramp voltage protocol, while the oocyte was bathed with a saline solution containing either of six distinct anions: Methanesulfonate‐(M), I‐,Br‐,Cl‐,F‐, or Thiocyanate‐(S). (B) Average value of the reversal potential from eight oocytes from two frogs. (C) Average relative permeability values of the distinct anions.
	Figure 5. Hypoosmotic challenge enhances ICl‐G1 and reveals a third type of current (ICl‐G3).(A) 1. Series of representative currents, obtained from an oocyte, 3 days after injection with Giardia′s mRNA, under the same protocol of stimulation, while bathed with a normal solution SBRS (left), then with a modified, isosmotic solution MBRS‐ISO (middle), then with a modified hypoosmotic solution (right). 2. Time course of the response to hypoosmotic challenge of mRNA‐injected oocytes. Alternate test pulses to +80 and −140 mV were given to a mRNA‐injected oocyte every 30 sec during 4 min. 3. IV relationships of currents obtained from control (upper) and mRNA‐injected (lower) oocytes while bathed with either SBRS, MBRS‐ISO, or MBRS‐HO. 4. Statistical comparison of the average slope conductance at −140 or +80 mV of control (upper) and mRNA‐injected (lower) oocytes while bathed with the three distinct media. Pairwise comparison (t‐test) indicates statistically significant difference (P < 0.05) for values before and after hypoosmotic challenge at −140 and +80 mV of mRNA‐injected but not of control oocytes. (B) 1. Representative series of currents of control (upper) and mRNA‐injected (lower) oocytes, bathed with an isoosmotic (upper set) or a hypoosmotic (lower set) solution, oocytes were stimulated with a prepulse of −80 mV (left) and 0 mV (middle) to obtain a difference, corresponding to ICl‐G1. It can be observed that hypoosmotic challenge enhances the magnitude of the difference (ICl‐G1) currents of mRNA‐injected but not from control oocytes. 2. IV relationship of the difference (ICl‐G1) current, before (red circles) and after (blue circles) of control (upper) and mRNA‐injected oocytes. (C) 1. Representative series of currents obtained from a control (upper row) and a mRNA‐injected (lower row) oocyte, in both cases oocytes were injected with BAPTA to inhibit outward (ICL‐G2) currents, and with a prepulse of 0 mV to minimize ICl‐G1 currents. A new type of current (ICl‐G3) is revealed by substracting currents under hypoosmotic and isoosmotic conditions recorded from mRNA‐injected oocytes, whereas those from control oocytes observed no difference currents. 2. IV relationships from recordings of control (upper) and mRNA‐injected (lower) oocytes under the distinct experimental conditions described above, the number of cases is the same as in figure (C.3). 3. Comparison of the slope conductances at +80 and −14 mV of control (upper) and mRNA‐injected (lower) oocytes under isosmotic and hypoosmotic conditions, as well as their difference. Statistical analysis (t‐test) indicates that the slope conductance at +80 mV of mRNA‐injected oocytes is significantly distinct from 0 (P < 0.01)
	Figure 6. Pharmacological properties of chloride currents induced by injection of mRNA of Giardia. The properties of the exogenous currents (IClG1‐G3) are shown in three rows and four columns. The first two columns (from left to right) show a representative example, before and after the addition of the blocker indicated in the second column, the third column shows the IV relationship before (black circles) and after (red circles) the addition of the blocker. The fourth column shows the effect of distinct chloride channel blockers on each type of currents. The results are expressed as % blocking, under the experimental conditions described in Results.

References [+] :

Ackerman, Hypotonicity activates a native chloride current in Xenopus oocytes. 1994, Pubmed, Xenbase