A Glial K/Cl Transporter Controls Neuronal Receptive Ending Shape by Chloride Inhibition of an rGC

Abstract

Neurons receive input from the outside world or from other neurons through neuronal receptive endings (NREs). Glia envelop NREs to create specialized microenvironments; however, glial functions at these sites are poorly understood. Here, we report a molecular mechanism by which glia control NRE shape and associated animal behavior. The C. elegans AMsh glial cell ensheathes the NREs of 12 neurons, including the thermosensory neuron AFD. KCC-3, a K/Cl transporter, localizes specifically to a glial microdomain surrounding AFD receptive ending microvilli, where it regulates K(+) and Cl(-) levels. We find that Cl(-) ions function as direct inhibitors of an NRE-localized receptor-guanylyl-cyclase, GCY-8, which synthesizes cyclic guanosine monophosphate (cGMP). High cGMP mediates the effects of glial KCC-3 on AFD shape by antagonizing the actin regulator WSP-1/NWASP. Components of this pathway are broadly expressed throughout the nervous system, suggesting that ionic regulation of the NRE microenvironment may be a conserved mechanism by which glia control neuron shape and function.

Figure 1. Glial KCC-3 controls AFD neuron receptive-ending shape

(A) AMsh glia ensheathes multiple neurons. AFD (orange) also has a cilium (blue). Boxed region magnified in Figure 7. (B) Histogram depicting AFD defects in indicated genotypes. Numbers, animals scored. Error bars, SEM. ***p<0.0002. For transgenics, three independent lines were scored. Green text, glia expression; red text, AFD expression. Second bar, non-transgenic siblings (C,D) Fluorescence images of wild-type (C) and kcc-3(ok228) (D) AFD microvilli. Scale bar, 1µm. Arrow, dendritic shaft. Arrowhead, microvilli. (E) kcc-3 gene structure, deletions used are indicated by red and blue bars. Sequences not included in WormBase annotation, orange. (F) Histogram details as in Figure 1B. Time, hours post mid L4 stage. (G, H) Thermotaxis behavior assays for indicated genotype raised at 15°C (G) and 25°C (H). Animals in (G), 24 hours post mid-L4 stage. Animals in (H), 96 hours post mid-L4 stage. N, Number of animals. See also Figure S1 and S2.

Figure 2. KCC-3 localizes to glial membranes surrounding AFD and regulates KCl homeostasis

(A,B) Fluorescence images depicting expression of indicated transcriptional reporters. Box, cells of interest. Circles + line in (B) indicate distinct cells. (C) Histogram details as in 1B and 1F. +, presence of kcc-3(ok228) allele. **p=0.009. (D, E) Arrow, AFD receptive-ending region. Asterisk, location of AWC cilium. (F, G) Histogram details as in 1B and 1F. **p<0.02. n.s., not significant compared to age-matched kcc-3(ok228). Hatched bars, animals with disorganized AFD microvilli.

Figure 3. The neuronal rGC GCY-8 functions downstream of glial KCC-3

(A) GCY-8 domain structure. (B) Histogram details as in 1B and 1F. *p=0.34. (C) Partial alignment of GCY-8. Red, conserved glycine mutated in gcy-8(ns335). (D,F,H) Fluorescence, TEM, and FIB-SEM reconstruction, respectively, of gcy-8(ns335) AFD receptive-ending. (E, G) TEM and FIB-SEM reconstruction, respectively, of wild-type AFD receptive-ending. (D) Arrow, dendritic shaft. Arrowhead, short microvilli. (E,F) Arrowheads, microvilli. Asterisk, dendritic shaft. Arrow, glial cytosol. Scale bar, 1µm. (I) Histogram details as in 1B. (J, K) Thermotaxis assays as in 1G. Animals raised at 15°C (J) or 25°C (K). (L) Histogram as in 1B. Hatched, animals with disorganized microvilli. See also Figure S3, S4 and Movies 1–4.

Figure 4. Excess GCY-8 and cGMP cause loss of AFD receptive-ending

Histogram details are as in Figure 1B. +, −, presence or absence, respectively, of gcy-8(ns335). Diagrams depict overexpressed GCY-8 protein. White-blue, weak-strong predicted cyclase activity (A). **p<0.02. n.s., not significant.

Figure 5. Chloride inhibits GCY-8 cyclase basal activity

(A) Western blot showing expression of indicated rGC proteins in HEK239T cells. (B) Histogram showing steady-state cGMP levels in HEK239T cells expressing the indicated constructs. Error bars, SEM of 3–4 experiments. ***p<0.0008. (C) cGMP measurements in membrane fractions of control HEK293T cells or GCY-8-expressing HEK293T cells. (D) Inhibition of GCY-8 membrane fraction guanylyl cyclase activity by chloride. Dotted lines, half-maximal effect. X-axis is plotted on a log₁₀ scale. (E, F) Histogram details as in Figure 1B. (F) Mutations engineered in full-length GCY-8 noted in color, see also Figure S5C.*=p<0.03 (G) Histogram details as in Figure 5C. Mutations noted as in Figure 5F. ***p=0.004; **p=0.01 (H) Details as in Figure 5D and 5F. Activity of each protein normalized to basal activity in 0mM NH4⁺Cl⁻ (dashed grey line). See also Figure S5.

Figure 6. The actin regulator WSP-1 regulates AFD shape downstream of GCY-8

(A–C) Histogram details as in Figure 1B. (A)+/−, presence or absence, respectively of PDE-1B over-expression (B) Gray, animals with extended microvilli. (C) Dotted, animals with slightly shorter, but not absent, microvilli. (D–F) Thermotaxis assays as in Figure 1G. 24 hours post mid-L4 stage animals raised at 15°C (D) or 25°C (E), and 96 hours post mid-L4 stage animals raised at 25°C (F). See also Figure S6 and S7.

Figure 7. Model for AFD neuron receptive-ending shape control by AMsh glia

Magnified view of boxed region in Figure 1A. Glial KCC-3 regulates chloride in the extracellular milieu around AFD receptive-ending. Chloride inhibits GCY-8 by binding the S(x)_nGPxC motif in its ECD. GCY-8, PDE-1 and PDE-5 control cGMP levels, which antagonizes the actin cytoskeleton through WSP-1.