A calcineurin homologous protein is required for sodium-proton exchange events in the C. elegans intestine

Abstract

Caenorhabditis elegans defecation is a rhythmic behavior, composed of three sequential muscle contractions, with a 50-s periodicity. The motor program is driven by oscillatory calcium signaling in the intestine. Proton fluxes, which require sodium-proton exchangers at the apical and basolateral intestinal membranes, parallel the intestinal calcium flux. These proton shifts are critical for defecation-associated muscle contraction, nutrient uptake, and longevity. How sodium-proton exchangers are activated in time with intestinal calcium oscillation is not known. The posterior body defecation contraction mutant (pbo-1) encodes a calcium-binding protein with homology to calcineurin homologous proteins, which are putative cofactors for mammalian sodium-proton exchangers. Loss of pbo-1 function results in a weakened defecation muscle contraction and a caloric restriction phenotype. Both of these phenotypes also arise from dysfunctions in pH regulation due to mutations in intestinal sodium-proton exchangers. Dynamic, in vivo imaging of intestinal proton flux in pbo-1 mutants using genetically encoded pH biosensors demonstrates that proton movements associated with these sodium-proton exchangers are significantly reduced. The basolateral acidification that signals the first defecation motor contraction is scant in the mutant compared with a normal animal. Luminal and cytoplasmic pH shifts are much reduced in the absence of PBO-1 compared with control animals. We conclude that pbo-1 is required for normal sodium-proton exchanger activity and may couple calcium and proton signaling events.

Fig. 1.

Characterization of posterior body contraction mutant 1 (pbo-1) motor program defects. A: defecation cycle in Caenorhabditis elegans (50). Worms execute the motor program approximately every 45–55 s while feeding. During the posterior body muscle contraction, the body muscles surrounding the intestine contract as a wave directed toward the anterior, forcing the contents of the gut forward. Approximately 3–5 s later, the anterior body muscles contract, propelling the gut contents backward (anterior body contraction). This is followed about a half-second later by an enteric muscle contraction that opens the anus and forcibly expels waste products. B: posterior body contraction strength. Contraction strength percentages normalized to wild-type samples (100%) are shown. Three cycles per animal were scored; n = 7 animals for each genotype except pbo-1(tm3716), where n = 5 [means ± SE: wild type 100.0 ± 9.1%; pbo-1(tm3716) 6.5 ± 2.5%, P < 0.001; pbo-1(sa7) 19.6 ± 3.0%, P < 0.001; pbo-4(ok583) 45.5 ± 6.9%, P < 0.001]. **P < 0.001 using an unpaired Student's t-test with unequal variance. C: mean percentage of posterior body contractions per cycle. Ten motor programs were scored per worm, 11 animals per genotype [means ± SE: wild type, 100.0 ± 0.0%; pbo-1(tm3716), 10.9 ± 4.6%, P < 0.0001; pbo-1(sa7), 87.3 ± 6.0%; pbo-1(sa7)/+, 100.0 ± 0.0%; pbo-1(tm3716)/+, 100.0 ± 0.0%; pbo-1(sa7)/pbo-1(tm3716), 0.9 ± 0.9%, P < 0.0001; pbo-1(tm3716)[Pvit-2:PBO-1], 100.0 ± 0.0%]. **P < 0.0001 using an unpaired Student's t-test with unequal variance. D: mean percentage of enteric muscle contractions per cycle. Ten motor programs were scored per worm, 11 animals per genotype [means ± SD, wild type, 98.2 ± 1.2%; pbo-1(tm3716), 94.6 ± 2.9%; pbo-1(sa7), 90.9 ± 2.5%, P < 0.05; pbo-1(sa7)/+, 99.1 ± 1.0%; pbo-1(tm3716)/+, 99.1 ± 1.0%; pbo-1(sa7)/pbo-1(tm3716), 100.0 ± 0.0%; pbo-1(tm3716)[Pvit-2:PBO-1], 85.5 ± 5.3%, P < 0.05]. *P < 0.05 using an unpaired Student's t-test with unequal variance. E–J: representative images of worms during a defecation motor program. Frames were taken from bright field movies. The first row of images shows the animal just before posterior body contraction. The second row displays the animal at maximal posterior body contraction. The lumen of the intestine has been shaded white. E: wild-type animal, just prior to contraction. F: pbo-1(tm3716), estimated precontraction. Since posterior body contractions are not readily visible in this mutant, the timing of the posterior body contraction was estimated relative to the onset of enteric muscle contraction. G: pbo-1(sa7), just before contraction. H: wild-type animal at maximal posterior body contraction. Arrows point to region of contraction. I: pbo-1(tm3716) at estimated maximal posterior body contraction. Since posterior body contractions are not readily visible in this mutant, the timing of the posterior body contraction was estimated relative to the enteric muscle contraction. J: pbo-1(sa7) at maximal posterior body contraction. Arrows point to small region of contraction.

Fig. 2.

Expression pattern of pbo-1. A–E: expression of green fluorescent protein (GFP) driven by the promoter of pbo-1, Ppbo-1::GFP. A: DIC image of an L2 stage animal. Scale bar equals 50 μm. Anterior is to the right. B: fluorescent image of the worm shown in A. Fluorescence is observed in the intestine of the larval animal, where it accumulates in the nuclei (seen as bright circles, arrows). Fluorescence is also visible in a number of neurons in the head and one neuronal process in the tail (indicated by arrowheads). C: DIC image of the head and anterior intestine of an adult animal. Scale bar equals 50 μm. D: fluorescence image of the animals shown in C. Expression is evident in the anterior intestine and many neurons. Fluorescence is found in neuronal processes, likely the anterior pharyngeal nerve ring and terminal bulb (arrows), as well as in the ventral cord (arrowheads). E: merged image of C and D. Arrows and arrowheads mark the same areas as in D.

Fig. 3.

Intestinal calcium dynamics in control, pbo-1, and nhx-2(RNAi) animals. A–D: mean change in fluorescence intensity of the calcium indicator, cameleon, for each genotype assayed. The means were determined from one representative cycle per worm. The error bars indicate SE. The y-axis is the ratio (R) of fluorescence emission relative to background emission (R₀). The x-axis represents time (in s). A: mean calcium change during approximately one defecation cycle period of the control, genotype = pha-1(e2123ts)III; unc-31(n422)IV; rnyEx001[Pnhx-2::YC61; pha-1(+)], n = 9. B: mean calcium change during approximately one defecation cycle period of pbo-1(tm3716), genotype = pbo-1(tm3716)III; rnyEx001[Pnhx-2::YC61; pha-1(+)], n = 9. C: mean calcium change during approximately one defecation cycle period of pbo-1(sa7), genotype = pbo-1(sa7)III; unc-31(n422)IV; rnyEx001[Pnhx-2::YC61; pha-1(+)], n = 10. D: mean calcium change during approximately one defecation cycle period of nhx-2(RNAi), genotype = pha-1(e2123ts)III; unc-31(n422)IV; rnyEx001[Pnhx-2::YC61; pha-1(+)], feed pRNAi-nhx-2, n = 6. E: graph with one sample trace for each genotype shown in A–D. ■, control genotype (A); ▴, pbo-1(tm3716) (B); ●, pbo-1(sa7) (C); ♦, nhx-2(RNAi) (D). F: mean amplitude of calcium change for genotypes in A–D. This mean incorporates every defecation cycle successfully recorded for each genotype. The total number of individual animals scored is listed above. Means ± SE are as follows: control, 0.26 ± 0.01; pbo-1(tm3716), 0.29 ± 0.01; pbo-1(sa7), 0.29 ± 0.04; nhx-2(RNAi), 0.33 ± 0.05.

Fig. 4.

Pseudocoelomic pH change in control, pbo-1 and pbo-4/nhx-7(ok583) animals. A–D: mean change in fluorescence intensity of the pseudocoelomic pH indicator, basolaterally localized pHluorin, for each genotype assayed. The means were determined from one representative cycle per worm. The error bars indicate SE. The y-axis is the approximate pH value. The x-axis represents time (in s). A: mean pseudocoelomic pH change during approximately one defecation cycle period of the control, genotype = pha-1(e2123ts)III; rnyEx009[Ppbo-4/nhx-7::PAT-3::pHluorin, pha-1(+)], n = 5. B: mean pseudocoelomic pH change during approximately one defecation cycle period of pbo-1(tm3716), genotype = pbo-1(tm3716)III; rnyEx009[Ppbo-4/nhx-7::PAT-3::pHluorin, pha-1(+)], n = 8. C: mean pseudocoelomic pH change during approximately one defecation cycle period of pbo-1(sa7), genotype=pbo-1(sa7); rnyEx009[Ppbo-4/nhx-7::PAT-3::pHluorin, pha-1(+)], n = 9. D: mean pseudocoelomic pH change during approximately one defecation cycle period of pbo-4/nhx-7(ok583), genotype = pbo-4/nhx-7(ok583)X; pha-1(e2123ts)III; rnyEx009[P pbo-4/nhx-7::PAT-3::pHluorin, pha-1(+)], n = 4. E: graph with one sample trace for each genotype shown in A–D. ■, control genotype (A); ▴, pbo-1(tm3716) (B); ●, pbo-1(sa7) (C); ▾, pbo-4(ok583) (D). F: mean amplitude of pseudocoelomic pH change for genotypes in A–D. This mean incorporates every defecation cycle successfully recorded for each genotype. The total number of individual animals scored is listed above. The means ± SE are as follows: control, 0.54 ± 0.07; pbo-1(tm3716), 0.15 ± 0.5; pbo-1(sa7), 0.11 ± 0.01, pbo-4/nhx-7(ok583), 0.14 ± 0.02. *P < 0.01 using an unpaired Student's t-test with unequal variance.

Fig. 5.

Luminal pH change in the intestines of control, pbo-1 and nhx-2(RNAi) animals. A–D: mean change in fluorescence intensity of the ingested luminal pH indicator, Oregon Green, for each genotype assayed. The means were determined from one representative cycle per worm. The error bars indicate SE. The y-axis is the approximate pH value. The x-axis represents time (in s). A: mean luminal pH change during approximately one defecation cycle period of the control, wild type/N2, n = 7. B: mean luminal pH change during approximately one defecation cycle period of pbo-1(tm3716), n = 8. C: mean luminal pH change during approximately one defecation cycle period of pbo-1(sa7), n = 9. D: mean luminal pH change during approximately one defecation cycle period of nhx-2(RNAi), n = 4. E: graph with one sample trace for each genotype shown in A–D. ■, control genotype (A); ▴, pbo-1(tm3716) (B); ●, pbo-1(sa7) (C); ♦, nhx-2(RNAi) (D). F: mean amplitude of luminal pH change for genotypes in A–D. This mean incorporates every defecation cycle successfully recorded for each genotype. The total number of individual animals scored is listed above. The means ± SE are as follows: control, 1.60 ± 0.11; pbo-1(tm3716), 0.60 ± 0.06; pbo-1(sa7), 0.50 ± 0.06, nhx-2(RNAi), 0.91 ± 0.12. *P < 0.01 and **P < 0.001 using an unpaired Student's t-test with unequal variance.

Fig. 6.

Cytoplasmic pH change in the intestines of control, pbo-1, and nhx-2(RNAi) animals. A–D: mean change in fluorescence intensity of the cytoplasmically localized pH indicator, cytoplasmic pHluorin, for each genotype assayed. The means were determined from one representative cycle per worm. The error bars indicate SE. The y-axis is the approximate pH value. The x-axis represents time (in s). A: mean cytoplasmic pH change during approximately one defecation cycle period of the control, genotype = pha-1(e2123ts)III; him-5(e1490)V; rnyEx006[Pnhx-2::pHluorin], n = 8. B: mean cytoplasmic pH change during approximately one defecation cycle period of pbo-1(tm3716), genotype = pbo-1(tm3716)III; rnyEx006[Pnhx-2::pHluorin], n = 11. C: mean cytoplasmic pH change during approximately one defecation cycle period of pbo-1(sa7), genotype = pbo-1(sa7)III; rnyEx006[Pnhx-2::pHluorin], n = 8. D: mean cytoplasmic pH change during approximately one defecation cycle period of nhx-2(RNAi), genotype = pha-1(e2123ts)III; him-5(e1490)V; rnyEx006[Pnhx-2::pHluorin], feed pRNAi-nhx-2, n = 6. E: graph with one sample trace for each genotype shown in A–D. ■, control genotype (A); ▴, pbo-1(tm3716) (B); ●, pbo-1(sa7) (C); ♦, nhx-2(RNAi) (D). F: mean amplitude of pH change for genotypes in A–D. This mean incorporates every defecation cycle successfully recorded for each genotype. The total number of individual animals scored is listed above. The means ± SE are as follows: control, 0.35 ± 0.03; pbo-1(tm3716), 0.15 ± 0.01; pbo-1(sa7), 0.18 ± 0.02; nhx-2(RNAi), 0.11 ± 0.01. *P < 0.01 and **P < 0.001 using an unpaired Student's t-test with unequal variance.

Fig. 7.

Fat stores in control, pbo-1, and nhx-2(RNAi) animals. A–D: fat contents were visualized by Oil Red-O staining. Images were taken at ×10 magnification, and signal appears as dark dots. Arrows point to the intestine and arrowheads point to the gonad. Scale bar equals 50 μm. A: wild-type Oil Red-O staining pattern, genotype = N2. B: pbo-1(tm3716) Oil Red-O staining pattern, genotype = pbo-1(tm3716)III. C: pbo-1(sa7) Oil Red-O staining pattern, genotype = pbo-1(sa7)III. D: nhx-2(RNAi) Oil Red-O staining pattern, genotype = pha-1(e2123ts)III.

Fig. 8.

A model of the signaling pathways governing the initiation of the C. elegans posterior body contraction. This schematic diagram shows select membrane transporters and electrolytes in the posterior intestine involved in signaling defecation behavior, highlighting the proposed role of PBO-1 as an NHX accessory protein. Every 45–55 s, a calcium wave is initiated by inositol 1,4,5-trisphosphate receptor (IP₃R) opening and resultant calcium release. The wave propagates, in a posterior-to-anterior fashion, through gap junctions containing the INX-16 protein. PBO-1 contributes to sodium-proton exchange activity at the apical and basolateral membranes. NHX-2 resides in the apical membrane and is physiologically coupled to nutrient uptake via OPT-2/PEPT-1-mediated dipeptide absorption. PBO-1 also contributes to proton efflux at the basolateral membrane via PBO-4/NHX-7, which signals the body wall muscles to contract through activation of the proton receptor PBO-5/PBO-6. This model suggests that PBO-1 may function to acutely regulate the exchange activity of NHX-2 and PBO-4/NHX-7 in response to calcium by conformational changes of the exchanger upon binding (A) and/or by regulating exchanger trafficking or membrane stabilization (B). Accordingly, in the diagram, active sodium-proton exchangers are bound by Ca²⁺-activated PBO-1; by contrast, those exchangers not bound by PBO-1 are inactive. ER, endoplasmic reticulum.