Localization of K⁺, H⁺, Na⁺ and Ca²⁺ fluxes to the excretory pore in Caenorhabditis elegans: application of scanning ion-selective microelectrodes

The importance of the nematode Caenorhabditis elegans Maupas 1900 as a model organism for studies of the physiological and molecular mechanisms of salt and water homeostasis has been highlighted in a recent review (Choe, 2013). As with other areas of C. elegans research, studies of excretory function are aided by the ease of culture, the availability of a fully-sequenced genome, low-cost access to mutant strains, the invariant lineage of somatic cells and the relative ease with which expression of specific genes can be silenced using RNA interference techniques. The excretory system of C. elegans consists of just three cells: an excretory cell, duct cell and pore cell. The large, H-shaped excretory cell forms two canals which run along much of the length of the worm. Ultrastructural studies show that the output of the excretory cell passes through the duct cell to an excretory pore, which opens to the outside of the animal just anterior to the pharynx (Nelson et al., 1983). Laser ablation of any of the three cells of the excretory system results in worm swelling and eventual mortality, implicating these cells in water homeostasis (Nelson and Riddle, 1984). The excretory cell is richly endowed with ion channels and transporters, including Cl⁻ channels, Na⁺/H⁺ exchangers, the vacuolar-type H⁺-ATPase and aquaporins (Choe, 2013). However, the drawbacks to the use of C. elegans in studies of ion excretion and osmoregulation are the thick cuticle and small size of the worms, greatly complicating the collection and analysis of the excretory fluids (Choe, 2013). The transport functions of the excretory cell have thus been largely unexplored because of the technical challenges inherent in studies of such a small organism.

In this paper, we describe a new method for analysis of ion efflux and influx by intact worms. Transport of ions into or out of worms bathed in saline produces gradients in ion concentration in the unstirred layer adjacent to the surface of the worm. These gradients can be quantified using the scanning ion-selective electrode technique (SIET), whereby voltages are recorded from an ion-selective microelectrode moved between two points within the unstirred layer. This method provides a means for precise localization of the sites of ion uptake and release.

Fig. 1 shows voltage differences recorded by ion-selective microelectrodes (K⁺, H⁺, Na⁺ and Ca²⁺) positioned near the surface of adult C. elegans. The length of each arrow in Fig. 1 indicates the voltage difference when the microelectrode was moved between a position within 3–5 μm of the surface of the worm and a second position 50 μm further away. Arrows pointing away from the worm indicate that the voltage was more positive at the inner limit, consistent with a higher cation concentration in the unstirred layer near the surface of the worm relative to the outer position.

For each of the four ions, a clear pattern of voltage gradients was apparent. The voltage difference was maximal on one side of the animal at a point just anterior to the junction of the pharynx and the intestine. The voltage differences indicated highly localized increases in the concentrations of K⁺, H⁺, Na⁺ and Ca²⁺ at a site consistent with the opening of the excretory pore, just anterior to the pharynx. Plots such as those in Fig. 1 were used to measure the distances from the anterior tip of the worm to the site of maximal voltage difference and from the tip to the readily identifiable junction of the pharynx and the intestine. For 38 worms, the mean distance to the site of the maximal voltage difference recorded by SIET was 133.9±5.1 μm (mean ± s.e.m.) and the distance to the junction of the pharynx and the intestine was 161.2±4.2 μm. When the distance to the site of maximal voltage difference was expressed as a proportion of the distance to the junction of the pharynx and the intestine, the values for K⁺ (0.86±0.03; N=11 worms), H⁺ (0.81±0.03, N=7), Na⁺ (0.83±0.05, N=13) and Ca²⁺ (0.80±0.03, N=7) were not significantly different. These results indicate that effluxes of all four cations peak at a single location on one side of the animal. There was no evidence of ion efflux from the mouth in any of the measurements, indicating that exposure to levamisole is not associated with leakage of the contents of the intestine or pharynx.

Voltage gradients at each site were converted into concentration gradients as described in the Materials and methods and ion fluxes (pmol cm⁻² s⁻¹) were then estimated from the measured voltage differences using the Fick equation. Plots of the fluxes for each ion as a function the distance anterior or posterior from the site of maximal ion flux are shown in Fig. 2. K⁺ effluxes were restricted to a narrow zone of <50 μm anterior or posterior to the site of maximal flux. Although this pattern was apparent for K⁺ fluxes for animals taken directly from the culture dishes, the maximal flux increased if worms were exposed to elevated K⁺ levels (Fig. 2A; 89 mmol l⁻¹) for periods of 40−150 min before SIET measurements. The high K⁺ conditions were produced by replacement of NaCl with KCl in M9 buffer. At distances of >100 μm posterior to the site of maximal efflux, K⁺ fluxes declined to small effluxes or, in a few worms, a small influx; the mean flux was 1.82±1.32 pmol cm⁻² s⁻¹ (N=10). H⁺ effluxes were restricted to a slightly wider zone, ~100 μm on either side of the site of maximal efflux (Fig. 2B). However, H⁺ effluxes of 1–3 pmol cm⁻² s⁻¹ were measured at distances of 200 μm or more posterior to the site of maximal efflux.

Localized Na⁺ fluxes were detected in less than half of the worms that were transferred directly from the culture medium (Fig. 2C). However, exposure of the worms to a high, but survivable (Choe and Strange, 2008), concentration of NaCl (400 mmol l⁻¹) for 1.5–3 h was associated with highly regionalized efflux, just anterior to the pharynx and consistent with the location of the excretory pore. However, there was also evidence of Na⁺ influx at regions more than ~100 μm posterior to the site of maximal efflux, consistent with inward transport of Na⁺ across the hypodermis at locations posterior to the excretory pore. The mean value of the influx was −20.4±2.7 pmol cm⁻² s⁻¹ (N=10 worms); values in worms taken directly from culture and those that had been preloaded with Na⁺ by exposure to 400 mmol l⁻¹ NaCl for 1.5–3 h did not differ and the results for both groups were therefore pooled. We also measured Na⁺ uptake across the hypodermis in animals bathed in a moderately hard reconstituted water (MHRW) (Khanna et al., 1997), which approximates the composition of soil water and contains 2.1 mmol l⁻¹ Na⁺. Measurements of worms taken directly from the culture medium indicated that Ca²⁺ fluxes were small and were not localized. However, effluxes of up to 25 pmol cm⁻² s⁻¹ were measured in worms that had been pre-incubated in saline containing 150 mmol l⁻¹ NaCl and 20 mmol l⁻¹ CaCl₂ for 1–2 h. Effluxes were found primarily within a zone of ~75 μm anterior or posterior to the site of maximal efflux (Fig. 2D). Efflux declined to a mean value of 0.63±0.29 pmol cm⁻² s⁻¹ at distances of >100 μm posterior to the site of maximal efflux.

Our SIET measurements thus provide direct evidence for localized efflux of K⁺, H⁺, Na⁺ and Ca²⁺ at a site on one side of the worm just anterior to the pharynx. Since the duct leading from the excretory cell opens through a pore located just anterior to the pharynx on the ventral surface of the animal (WormAtlas, www.wormatlas.org), our working hypothesis is that the effluxes described in the Results are the output of the excretory cell. These measurements thus provide the first direct evidence for ion excretion from the excretory cell of C. elegans. Previous studies have inferred the contributions of the cell to ionoregulation by worm swelling or mortality in response to variations in bathing medium ionic milieu when the functioning of the excretory cell was impaired by laser ablation (Nelson and Riddle, 1984).

In addition to the finding that ion effluxes are highly localized, we have also shown that efflux is enhanced when the worms are exposed to high concentrations of K⁺, Na⁺ or Ca²⁺ prior to measurements with SIET. Our working hypothesis is that the excretory cell contributes to homeostasis through excretion of these ions when they are present to excess. K⁺ effluxes were clearly evident in worms taken directly from culture; we suggest that this reflects the high K⁺ content of the bacterial food of the worms (Richey et al., 1987). Although the efflux of Na⁺ efflux is localized to a site consistent with the location of the excretory pore, Na⁺ influx occurs across regions of the worm posterior to the pore, suggesting that the hypodermis is a site of Na⁺ uptake from the bathing medium. C. elegans is a soil nematode which may be exposed to Na⁺-poor soil water. Under such conditions uptake of Na⁺ across the hypodermis may be an important contributor to Na⁺ homeostasis. The hypodermis is known to contain Na⁺ transporters such as the Na⁺:H⁺ exchanger homolog NHX-3 (Nehrke and Melvin, 2002) and SIET measurements may be of use in future experiments to characterize the functional roles of such transporters. We detected Ca²⁺ effluxes localized to the excretory pore only after the worms had been exposed to high levels of Ca²⁺ prior to SIET measurements. Previous studies have shown that a member of the TRPM protein family, GTL-2, acts within the C. elegans excretory cell to mediate the excretion of excess magnesium (Teramoto et al., 2010), but much less is known of the transporters involved in excretion of excess Ca²⁺.

In summary, we believe that SIET provides a means to measure both excretion of ions from the excretory system as well as ion transport across the hypodermis. The small size of C. elegans has been a barrier to physiological measurements of salt and water homeostasis. We suggest that ion-selective microelectrodes and SIET provide a means of circumventing this barrier, allowing subsequent studies to exploit the powerful molecular genetic tools available for studies of water and ion homeostasis in this model organism.

The N2 strain of C. elegans was obtained from the Caenorhabditis Genetics Center at the University of Minnesota and was reared on E. coli OP50 on nematode growth medium (NGM) agar plates. Adult worms were used for all experiments. Ion fluxes out of, or into, the worms were determined using SIET. An orthogonal array of computer-controlled stepper motors fitted to a set of translator stages allow the ion-selective microelectrode to be positioned in three dimensions with submicrometer accuracy and repeatability. The microelectrode was first positioned 3–5 μm from the surface of the worm. After a wait period of 4–5 s to allow re-establishment of ion concentration gradients that had been disturbed by microelectrode movement, the microelectrode was repositioned 50 μm further out from the worm, along a line perpendicular to the long axis of the worm. The voltage at the outer site was then recorded after a further wait period, and the voltage difference between the inner and outer limits of microelectrode excursion was calculated. Hardware used for SIET measurements was obtained from Applicable Electronics (Forestdale, MA, USA) and software for control of microelectrode positioning and data acquisition was obtained from ScienceWares (Falmouth, MA, USA). The application of SIET and the construction and calibration of ion-selective microelectrodes for K⁺, Na⁺, H⁺ and Ca²⁺ have been described previously (Donini and O'Donnell, 2005; Jayakannan et al., 2011).

To permit SIET measurements of ion concentration gradients in the unstirred layer near the surface of the worm, locomotion was inhibited by inclusion of the cholinergic agonist levamisole (0.5 mmol l⁻¹) in the bathing saline. Levamisole induces rigid paralysis by overstimulation of the body wall muscle. Approximately 50% of the worms had stopped moving and could be used for experiments within 30 min of levamisole treatment. In most animals, small movements of the mouth or pharyngeal pumping were observed throughout the period of exposure to levamisole.

Detection of localized changes in ion concentration by SIET was facilitated by bathing the worms in saline containing 150 mmol l⁻¹ N-methyl-D-glucamine (NMDG) chloride, which does not interfere with Na⁺-or K⁺-selective microelectrodes. The saline also contained 1 mmol l⁻¹ MgCl₂ and a low concentration of the ion of interest (0.15 mmol l⁻¹ K⁺, 0.15–3 mmol l⁻¹ Na⁺, 0.01 mmol l⁻¹ Ca²⁺, 10⁻⁷ mol l⁻¹ H⁺). Saline for H⁺, Na⁺ and K⁺ measurements contained 1.8 mmol l⁻¹ CaCl₂. Saline osmolality is close to that of M9 buffer (~300 mosmol kg⁻¹) commonly used for C. elegans. Worms continued to move for >12 h in saline in the absence of levamisole. Saline was buffered with 5 mmol l⁻¹ Hepes for Na⁺ and K⁺ measurements. Buffering capacity was reduced in H⁺ flux measurements by lowering the Hepes concentration to 1 mmol l⁻¹. H⁺ fluxes were corrected for the effects of the buffer using equations published previously (Messerli et al., 2006). PIPES was used as a buffer for Ca²⁺ measurements because it does not form complexes with divalent metals such as Ca²⁺ (Yu et al., 1997). In all cases, worms were rinsed in 150 mmol l⁻¹ NMDG and 5 mmol l⁻¹ Hepes to remove bacteria and adherent fluids before transfer to the Petri dish used for SIET measurements.

We are grateful to Siavash Amon for assistance with worm culture.