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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6264fjev1 20/14/2606    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Qian, H. Articles by Robertson, A. P. Search for Related Content PubMed PubMed Citation Articles by Qian, H. Articles by Robertson, A. P.

Pharmacology of N-, L-, and B-subtypes of nematode nAChR resolved at the single-channel level in Ascaris suum

Department of Biomedical Sciences, Iowa State University, Ames, Iowa, USA

²Correspondence: Department of Biomedical Sciences, #2008, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011-1250, USA. E-mail: rjmartin{at}iastate.edu

SPECIFIC AIMS

Previous contraction studies on the parasitic nematode Ascaris suum demonstrated that cholinergic anthelmintics can activate three (N-, L-, and B-) subtypes of receptors in muscle and that the L-subtype decreases with resistance to levamisole, a cholinergic anthelmintic. We examined the action of levamisole under patch clamp to determine if the three receptor channels could be separated and characterized pharmacologically.

Principle Findings
1. Levamisole activates three channels with a different conductance in body muscle of Ascaris
When levamisole (30 µM) was used as the agonist in the pipette solution, single-channel currents with a conductance of 18–53 pS and mean open times of 0.2–2.0 ms were observed in Ascaris body muscle. In each patch, levamisole activated up to three populations of channel currents that were clearly separated by differences in their conductance. Figure 1 illustrates examples of the small, intermediate, and large channel currents that were observed at + 75 mV in a single patch recording. In all experiments where three or more voltages could be tested between + 75 mV and –75mV, it was found that the current-voltage relationships of the channels were linear; therefore, conductance was determined by linear regression. In Fig. 1B , the channels had a conductance of 26 pS, 36 pS, and 48 pS.

View larger version (25K): [in this window] [in a new window]   Figure 1. Small, intermediate, and large channel nAChR current amplitudes present in a single inside-out patch activated by 30 µM levamisole. A) Single-channel openings from single patch recording at + 75 mV, showing examples of 3 separate channel currents: O1, O2, and O3 are the open levels, and C is closed level. B) Current amplitude histogram of patch recording best fitted by sum of 3 Gaussian distributions: peaks are 2.12 ± 1.47, 2.84 ± 0.05, and 3.83 ± 0.01 pA (mean±SE) and have conductances of 26, 36, and 48 pS .

We recorded levamisole-activated currents from 58 channels in 38 patches: 4 patches showed the 3 channels in the same patch. To determine the conductance ranges of the three populations, we determined the 95% confidence intervals of the small, intermediate, and large conductance populations from the four patches with the three channels. The confidence limits obtained from the four patches for the small channels were 17.8 and 26.3 pS; the limits for the intermediate channels were 26.5 and 38.9 pS; the limits for the largechannels were 36.6 and 49.3 pS. For convenience, we refer to the small channel as G₂₅, the intermediate channel as G₃₅, and the large channel as G₄₅. We used the confidence intervals to sort channels into the G₂₅, G₃₅, and G₄₅ groups to determine their abundance: two channels with a conductance between 36.6 and 38.9 pS could not be assigned to a specific group and were excluded from further analysis.

2. Levamisole preferentially activated G₃₅ channels
After sorting this way, we found that levamisole activated more G₃₅ channels than the other subtypes: 27% of the channels were G₂₅, 39% were G₃₅, and 34% were G₄₅. To estimate the proportion of each subtype present using another approach, we fitted the conductance histogram of all the channels activated by levamisole to the sum of three Gaussian distributions. Both analytical approaches separated three channel subtypes and showed that levamisole activated more G₃₅ channels than the other receptor channel subtypes.

3. Bephenium activates only G₃₅ and G₄₅ channels
When levamisole in the pipette solution was replaced by 1 or 10 µM bephenium (another cholinomimetic anthelmintic), only two channel types, G₃₅ and G₄₅, were observed in the patches. Bephenium did not activate the G₂₅ channels. Using the same approach that we used for the levamisole-activated channel currents, we evaluated the 95% confidence intervals for the 26 G₃₅ and G₄₅ channels recorded from 19 patches. We found that the G₃₅ channel had a 95% confidence interval of 31.6–40.2 pS and the G₄₅ channel had a 95% confidence interval of 41.6–47.1 pS. The means and the conductance ranges of the two bephenium-activated populations were comparable to those of the G₃₅ and G₄₅ channels activated by levamisole.

4. Bephenium activated more G₄₅ than G₃₅ channels
When 1 µM bephenium was the agonist, 38% of the channel population was G₃₅. Thus, bephenium activated more G₄₅ channels than G₃₅ channels, suggesting that these channels represent the B-subtype of receptors.

5. Different subtypes have different mean open times
With levamisole as the agonist, the G₂₅ population had a mean open time of 0.6 ± 0.1 ms (mean±SE; n=14); the mean open time of the G₃₅ population was 0.8 ± 0.1 ms (mean±SE; n=18); and the mean open time of the G₄₅ population was (1.2±0.1 ms mean±SE; n=17). The mean open time of the G₃₅ channels activated by 1 µM bephenium was 1.1 ± 0.2 ms (mean±SE; n=7); the mean open time of the G45 population was 2.4 ± 0.2 ms (mean±SE; n=11). The differences between the mean open times of the different conductance populations illustrate that the channel populations belong to separable, distinctive subtypes.

6. Bath application of paraherquamide inhibits the opening of nAChRs in inside-out patches
Our earlier muscle contraction experiments have demonstrated that paraherquamide is a competitive antagonists. Since paraherquamide passes through membranes, we were able to measure the antagonism and used paired recordings of the patch-Popen to control for effects of solution change. The inhibitory effect of paraherquamide was dose dependent and described by the logistic function with an IC₅₀ of 3.1 µM. We found no effect of paraherquamide on the amplitudes of any of the channel subtypes, nor did it decrease the mean open times of the channel subtypes: it only reduced P-open. The lack of effect on amplitude and open time is consistent with the competitive mode of action of paraherquamide.

7. Inhibitory effects of paraherquamide and 2-desoxoparaherquamide depends on the receptor subtype
We tested the inhibitory effects of 1 µM paraherquamide and 3 µM 2-desoxoparaherquamide on the Popen of the G₂₅, G₃₅, and G₄₅ channels (Fig. 2 ). Paraherquamide had no effect on the G₂₅ population but had a significant inhibitory effect on the G₃₅ and G₄₅ channels. The bigger effect on G₃₅ and G₄₅ channels is consistent with these channels belonging to the L- and B-subtypes because paraherquamide is a more potent antagonist of these two subtypes than the N-subtype in contraction studies. We also used 10 µM bephenium to activate the G₃₅ and G₄₅ channels without activating the G₂₅ channels. 2-Desoxoparaherquamide had a significantly greater inhibitory effect on the G₄₅ population than the G₃₅ population, an effect consistent with the G₄₅ channels being the B-subtype and the G₃₅ channels being the L-subtype.

View larger version (16K): [in this window] [in a new window]   Figure 2. Inhibitory effects of paraherquamide and 2-desoxyparaherquamide on G25, G35, and G45 are statistically different. Left) 1 µM paraherquamide had a greater effect on normalized Popen (NPo) mean ± SE of G35 channels (67.7±10.0% inhibition P<0.05, n=5) and G45 channels (68.2±7.0% inhibition P<0.05, n=5 and) than on the G25 channels (106±25.45, n=5); G25, G35, and G45 channels were activated by 30 µM levamisole. Right) 3 µM 2-desoxyparaherquamide had a significantly greater effect on the normalized Popen of 10 µM bephenium-activated G35 channels (36.4±11.4% inhibition, P<0.05, n=4) and G45 channels (73.3±11.4% inhibition, P<0.05, n=5) activated by 10 µM bephenium.

CONCLUSIONS AND SIGNIFICANCE

1.6 Billion people throughout the world carry ascariasis and hookworm infections. Approximately one-third of the world’s population is suffering from the effects of intestinal nematode parasites, causing low growth-rates in infants, ill-thrift, diarrhea, and in 2% of cases, loss of life. Levamisole and related drugs (pyrantel and oxantel) are particularly desirable for medication, because they have a rapid therapeutic effect within 2 h of administration. Consequently, these drugs are frequently used to combat human intestinal nematode parasites. The continued use of all anthelmintics, including levamisole, is expected to increase the level of resistance. Cure rates are often <100% and resistance of human parasites to pyrantel has been described previously. Because development of novel anthelmintics is very limited, it is important to understand details of modes of action of existing anthelmintics to determine methods of enhancing their efficacy and to develop approaches that will counter resistance. We have focused on levamisole and related drugs.

Comparison of levamisole and bephenium activated channel currents under patch-clamp separated N- (G₂₅), L- (G₃₅), and B- (G₄₅) subtypes in A. suum with different pharmacological sensitivities and channel conductance and mean open times (Fig. 3 ). Before we began our work on the novel antibiotic anthelmintic paraherquamide, its action at the single-channel level was not known. We described how paraherquamide, and the 2-desoxy derivative, separate N-, L-, and B-subtypes and have different selectivity. The findings of this study will inform the approach for treatment (for increasing efficacy and for managing drug resistance) of a significant human and animal health problems. For example it may be better to combine some of the cholinergic anthelmintics (pyrantel: L-subtype and oxantel: B-subtype) to increase the spectrum of action and to counter resistance. Future therapeutic use and resistance studies of cholinergic anthelmintics should take into account the heterogeneous nature of the nAChR population in parasitic nematodes and the different selectivity of the cholinergic anthelmintics.

View larger version (23K): [in this window] [in a new window]   Figure 3. Summary diagram. Levamisole (30 µM) activated 3 subtypes of nAChR channel currents in patch recordings from Ascaris muscle. The 3 types were small (G25: N-type, mean conductance 22 pS, mean open time 0.6 ms, not very sensitive to paraherquamide); intermediate (G35: L-type, mean conductance 33 pS, mean open time 0.8 ms, sensitive to paraherquamide (PHQ), less sensitive to desoxy-paraherquamide 2DPHQ); and large (G45: B-type mean conductance 45 pS, mean open time 1.2 ms, sensitive to paraherquamide and to desoxy-paraherquamide) conductance channels.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6264fje

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