SER-7, a Caenorhabditis elegans 5-HT7-like receptor, is essential for the 5-HT stimulation of pharyngeal pumping and egg laying

Abstract

Serotonin (5-HT) stimulates both pharyngeal pumping and egg laying in Caenorhabditis elegans. Four distinct 5-HT receptors have been partially characterized, but little is known about their function in vivo. SER-7 exhibits most sequence identity to the mammalian 5-HT7 receptors and couples to a stimulation of adenyl cyclase when expressed in COS-7 cells. However, many 5-HT7-specific agonists have low affinity for SER-7. 5-HT fails to stimulate pharyngeal pumping and the firing of the MC motorneurons in animals containing the putative ser-7(tm1325) and ser-7(tm1728) null alleles. In addition, although pumping on bacteria is upregulated in ser-7(tm1325) animals, pumping is more irregular. A similar failure to maintain "fast pumping" on bacteria also was observed in ser-1(ok345) and tph-1(mg280) animals that contain putative null alleles of a 5-HT2-like receptor and tryptophan hydroxylase, respectively, suggesting that serotonergic signaling, although not essential for the upregulation of pumping on bacteria, "fine tunes" the process. 5-HT also fails to stimulate egg laying in ser-7(tm1325), ser-1(ok345), and ser-7(tm1325) ser-1(ok345) animals, but only the ser-7 ser-1 double mutants exhibit an Egl phenotype. All of the SER-7 mutant phenotypes are rescued by the expression of full-length ser-7gfp translational fusions. ser-7gfp is expressed in several pharyngeal neurons, including the MC, M2, M3, M4, and M5, and in vulval muscle. Interestingly, 5-HT inhibits egg laying and pharyngeal pumping in ser-7 null mutants and the 5-HT inhibition of egg laying, but not pumping, is abolished in ser-7(tm1325);ser-4(ok512) double mutants. Taken together, these results suggest that SER-7 is essential for the 5-HT stimulation of both egg laying and pharyngeal pumping, but that other signaling pathways can probably fulfill similar roles in vivo.

Figure 1.

Genomic structure of ser-7 and characterization of ser-1 and ser-7 rescue constructs. (A) Exons are shown as open boxes, introns as a single line, and 5′- and 3′-UTRs are shaded. The positions of GFP in the ser-7∷gfp and ser-1∷gfp translational fusions are illustrated, as are the positions of the deletions in ser-7(tm1325), ser-7(tm1728) and ser-1(ok345) animals. The name of the transgenic line created with each construct is shown in parentheses. (B) Predicted membrane topology of SER-7, showing the position of GFP in the third-intracellular loop. GFP is located after residue R₂₅₁. Arrows indicate mutations in SER-7B_E314A/K316AI3GFP.

Figure 2.

The insertion of GFP into the SER-7B third-intracellular loop has no effect on G-protein coupling. Native SER-7B (○), SER-7BI3GFP (•), and SER-7B_E314A/K316AI3GFP (▪), were transiently expressed in COS-7 cells and EC₅₀s for 5-HT were determined, as described in materials and methods. 5-HT did not alter cAMP levels in untransfected COS-7 cells (□).

Figure 3.

SER-7 is essential for 5-HT-stimulated pharyngeal pumping. Pharyngeal pumping was assayed in the presence of E. coli OP50 and/or 13 mM 5-HT, as described in materials and methods, in N2's or animals carrying the ser-7 null alleles (tm1325 or tm1728), the ser-1 null allele (ok345), the ser-4 null allele (ok512), or in ser-7(tm1325);ser-4(ok512) or ser-7(tm1325) ser-1(ok345) double mutants. In addition, ser-7(tm1325) animals were rescued with a full-length ser-7∷gfp translational fusion [ser-7(tm1325)(+)]. *, different from N2 in the presence of bacteria (P < 0.005); †, different from N2 in the presence of 5-HT (P < 0.0005); ‡, different from N2 in the presence of 5-HT and bacteria (P < 0.0005). n = 30 for each strain.

Figure 4.

Serotonin receptor and synthesis mutants exhibit more variation in rates of pharyngeal pumping on bacteria than N2's. Pharyngeal pumping (pumps/10 sec) on bacteria was measured in individual animals every 30 sec for 10 min, as described in materials and methods. Pumping rates/10 sec from at least 7 animals were pooled and plotted as histograms for each strain. (A–G) N2, ser-7(tm1325), ser-7(tm1325)(+)(erEx1517), ser-1(ok345), ser-1(ok345)(+)(erEx1617), ser-7(tm1325) ser-1(ok345), and tph-1(mg280) animals were examined. Inset on each histogram shows the mean pumping rate ± SEM and the n for each strain. *, mean pumping rate/10 sec different from N2 by T-test (P < 0.0005); †, distribution of pumping rates different from N2 by Kolmogorov-Smirnov test (P < 0.005). The vertical black line on each graph represents 43 pumps/10 sec, the mean value for N2's.

Figure 5.

Expression of the full-length ser-7∷gfp translational fusion with sequence coding for GFP inserted into the predicted SER-7 third-intracellular loop is limited to a number of pharyngeal neurons and vulval muscle. The ser-7∷gfp PCR fusion was injected into ser-7(tm1325) animals and the transgenic worms (erEx1517) were examined for fluorescence using a confocal microscope. (A) Fluorescence from SER-7∷GFP in a subset of pharyngeal neurons. (B) SER-7∷GFP fluorescence in vulval muscle. (Left) GFP fluorescence in the vulval muscle and (right) an overlay of the GFP stack and a single DIC image showing the vulval opening.

Figure 6.

EPGs of N2, ser-7(tm1325) and ser-7(tm1325)(+) (erEx1517) animals. (A) EPGs were recorded in the presence of 13 mM 5-HT, as described in materials and methods. Asterisk indicates an E1 spike. Most N2's exhibit two E-phase transients (E1 and E2). In contrast, most (>95%) of the pumps of the ser-7(tm1325) animals lack the E1 transient, which has been previously been associated with MC activity (Raizen et al. 1995). (Inset) A few (<5%) of the traces from ser-7(tm1325) animals show an early E-phase transient (marked by the arrow) preceding the large E2 transient. (B) Histograms comparing the number of traces exhibiting an E1-phase transient in N2, ser-7(tm1325), and ser-7(tm1325)(+) (erEx1517) animals. Data from 200 traces from at least 10 different animals from each strain were examined.

Figure 7.

SER-7 is essential for the 5-HT stimulation of egg laying. Synchronized animals were placed on NGM plates in the presence of 13 mM 5-HT and/or E. coli OP50 and eggs laid were counted after 1hr, as described in materials and methods. N2's or animals carrying the ser-7 null alleles (tm1325 or tm1728), the ser-1 null allele (ok345), the ser-4 null allele (ok512) or in ser-7(tm1325);ser-4(ok512) or ser-7(tm1325) ser-1(ok345) double mutants were examined. In addition, ser-7(tm1325) animals were rescued with a full-length ser-7∷gfp translational fusion (ser-7(tm1325)(+). *, mean eggs laid/animal different from N2 in the presence of 5-HT (P < 0.005); **, mean eggs laid/animal different from N2 in the presence of 5-HT (P < 0.05); †, mean eggs laid/animal different from N2 in the presence of 5-HT and bacteria (P < 0.005). n = 45 for each strain.