Differential regulation of TRPM channels governs electrolyte homeostasis in the C. elegans intestine.

The transient receptor potential (TRP) channels are implicated in various cellular processes, including sensory signal transduction and electrolyte homeostasis. We show here that the GTL-1 and GON-2 TRPM channels regulate electrolyte homeostasis in the C. elegans intestine. GON-2 is responsible for a large outwardly rectifying current of intestinal cells, and its activity is tightly regulated by intracellular Mg(2+) levels, while GTL-1 mainly contributes to appropriate Mg(2+) responsiveness of the outwardly rectifying current. We also used nickel cytotoxicity to study the function of these channels. Both GON-2 and GTL-1 are necessary for intestinal uptake of nickel, but GTL-1 is continuously active while GON-2 is inactivated at higher Mg(2+) levels. This type of differential regulation of intestinal electrolyte absorption ensures a constant supply of electrolytes through GTL-1, while occasional bursts of GON-2 activity allow rapid return to normal electrolyte concentrations following physiological perturbations.

A calcium wave mediated by gap junctions coordinates a rhythmic behavior in C. elegans.

Intercellular calcium waves can be observed in adult tissues, but whether they are instructive, permissive, or even required for behavior is predominantly unknown. In the nematode Caenorhabditis elegans, a periodic calcium spike in a pacemaker cell initiates a calcium wave in the intestine. The calcium wave is followed by three muscle contractions that comprise the defecation motor program. Normal wave propagation requires the pannexin gap-junction subunit INX-16 at the interfaces of the intestinal cells. In the absence of this gap-junction subunit, calcium waves are frequently absent. The remaining waves are slow, initiate at abnormal locations, or travel in the opposite direction. Abnormal waves are associated with parallel effects in the first step of the motor program: The contractions of the overlying muscles fail to propagate beyond the pacemaker cell, are slow, initiate in abnormal locations, or are reversed. Moreover, the last two motor steps are predominantly absent. Finally, the absence of this gap-junction subunit also affects the reliability of the pacemaker cell; cycle timing is often irregular. These data demonstrate that pannexin gap junctions propagate calcium waves in the C. elegans intestine. The calcium waves instruct the motor steps and regulate the pacemaker cell's authority and reliability.

The non-neuronal syntaxin SYN-1 regulates defecation behavior and neural activity in C. elegans through interaction with the Munc13-like protein AEX-1.

We have previously shown that the AEX-1 protein, which is expressed in postsynaptic muscles, retrogradely regulates presynaptic neural activity at the Caenorhabditis elegans neuromuscular junctions. AEX-1 is similar to vertebrate Munc13-4 protein, suggesting a function for vesicle exocytosis from a kind of cells. Compared to emerging evidences of the role of Munc13 proteins in synaptic vesicle release, however, the precise mechanism for vesicle exocytosis by AEX-1 and Munc13-4 is little understood. Here we have identified SYN-1 as a candidate molecule of AEX-1-dependent vesicle exocytosis from non-neuronal cells. The syn-1 gene encodes a C. elegans syntaxin, which is distantly related to the neuronal syntaxin UNC-64. The syn-1 gene is predominantly expressed in non-neuronal tissues and genetically interacts with aex-1 for presynaptic activity. However, the two proteins did not interact physically in our yeast two-hybrid system and mutational SYN-1 did not bypass the requirement of AEX-1 for the behavioral defects in aex-1 mutants, whereas mutant UNC-64 does in unc-13 mutants. These results suggest that a novel molecular interaction between the AEX-1 and syntaxin may regulate vesicle exocytosis for retrograde signal release.

Na+/K+ ATPase regulates the expression and localization of acetylcholine receptors in a pump activity-independent manner.

Na+/K+ ATPase is a plasma membrane-localized sodium pump that maintains the ion gradients between the extracellular and intracellular environments, which in turn controls the cellular resting membrane potential.Recent evidence suggests that the pump is also localized at synapses and regulates synaptic efficacy.However, its precise function at the synapse is unknown. Here we show that two mutations in the alpha subunit of the eat-6 Na+/K+ ATPase in Caenorhabditis elegans dramatically increase the sensitivity to acetylcholine(Ach) agonists and alter the localization of nicotinic Ach receptors at the neuromuscular junction (NMJ).These defects can be rescued by mutated EAT-6 proteins which lack its pump activity, suggesting the presence of a novel function for Ach signaling. The Na+/K+ ATPase accumulates at postsynaptic sites and appears to surround Ach receptors to maintain rigid clusters at the NMJ. Our findings suggest a pump activity-independent, allele-specific role for Na+/K+ ATPase on postsynaptic organization and synaptic efficacy.

Regulation of retrograde signaling at neuromuscular junctions by the novel C2 domain protein AEX-1.

Retrograde signaling from postsynaptic cells to presynaptic neurons is essential for regulation of synaptic development, maintenance, and plasticity. Here we report that the novel protein AEX-1 controls retrograde signaling at neuromuscular junctions in C. elegans. aex-1 mutants show neural defects including reduced presynaptic activity and abnormal localization of the synaptic vesicle fusion protein UNC-13. Muscle-specific AEX-1 expression rescues these defects but neuron-specific expression does not. AEX-1 has an UNC-13 homologous domain and appears to regulate exocytosis in muscles. This retrograde signaling requires prohormone-convertase function in muscles, suggesting that a peptide is the retrograde signal. This signal regulates synaptic vesicle release via the EGL-30 Gq(alpha) protein at presynaptic terminals.

UNC-31 (CAPS) is required for dense-core vesicle but not synaptic vesicle exocytosis in Caenorhabditis elegans.

Previous studies indicated that CAPS (calcium-dependent activator protein for secretion) functions as an essential component for the Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells. However, recent mouse knock-out studies suggested an alternative role in the vesicular uptake or storage of catecholamines. To genetically assess the functional role of CAPS, we characterized the sole Caenorhabditis elegans CAPS ortholog UNC-31 (uncoordinated family member) and determined its role in dense-core vesicle-mediated peptide secretion and in synaptic vesicle recycling. Novel assays for dense-core vesicle exocytosis were developed by expressing a prepro-atrial natriuretic factor-green fluorescent protein fusion protein in C. elegans. unc-31 mutants exhibited reduced peptide release in vivo and lacked evoked peptide release in cultured neurons. In contrast, cultured neurons from unc-31 mutants exhibited normal stimulated synaptic vesicle recycling measured by FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)hexatrienyl)pyridinium dibromide] dye uptake. Conversely, UNC-13, which exhibits sequence homology to CAPS/UNC-31, was found to be essential for synaptic vesicle but not dense-core vesicle exocytosis. These findings indicate that CAPS/UNC-31 function is not restricted to catecholaminergic vesicles but is generally required for and specific to dense-core vesicle exocytosis. Our results suggest that CAPS/UNC-31 and UNC-13 serve parallel and dedicated roles in dense-core vesicle and synaptic vesicle exocytosis, respectively, in the C. elegans nervous system.

Intestinal calcium waves coordinate a behavioral motor program in C. elegans.

Periodic behavioral motor patterns are normally controlled by neural circuits, such as central pattern generators. We here report a novel mechanism of motor pattern generation by non-neural cells. The defecation motor program in Caenorhabditis elegans consists of three stereotyped motor steps with precise timing and this behavior has been studied as a model system of a ultradian biological clock [J.H. Thomas, Genetic analysis of defecation in C. elegans, Genetics 124 (1990) 855-872; D.W. Liu, J.H. Thomas, Regulation of a periodic motor program in C. elegans, J. Neurosci. 14 (1994) 1953-1962; K. Iwasaki, D.W. Liu, J.H. Thomas, Genes that control a temperature-compensated ultradian clock in Caenorhabditis elegans, Proc. Natl. Acad. Sci. USA 92 (1995), 10317-10321]. It was previously implied that the inositol-1,4,5-trisphosphate (IP3) receptor in the intestine was necessary for this periodic behavior [P. Dal Santo, M.A. Logan, A.D. Chisholm, E.M. Jorgensen, The inositol trisphosphate receptor regulates a 50s behavioral rhythm in C. elegans, Cell 98 (1999) 757-767]. Therefore, we developed a new assay system to study a relationship between this behavioral timing and intestinal Ca(2+) dynamics. Using this assay system, we found that the timing between the first and second motor steps is coordinated by intercellular Ca(2+)-wave propagation in the intestine. Lack of the Ca(2+)-wave propagation correlated with no coordination of the motor steps in the CaMKII mutant. Also, when the Ca(2+)-wave propagation was blocked by the IP3 receptor inhibitor heparin at the mid-intestine in wild type, the second/third motor steps were eliminated, which phenocopied ablation of the motor neurons AVL and DVB. These observations suggest that an intestinal Ca(2+)-wave propagation governs the timing of neural activities that controls specific behavioral patterns in C. elegans.

Time and Costs of Insulin Treatment in the Care of Newly Registered Type 2 Diabetes Patients in Diabetes Clinics Across Japan (JDDM 22).

AIMS: To study the time and costs of insulin treatment of newly registered outpatients with Type 2 diabetes mellitus (T2DM). METHODS: In total, 355 patients with T2DM were registered on their first visit to one of 11 diabetes clinics across Japan. Of these, 313 were not being treated with insulin (the non-insulin group), whereas 42 were (the insulin group). In the insulin group, 26 were already on insulin at the first visit, whereas 16 were started on insulin after their first visit. The time and costs involved in the care were recorded over the following 5 months. RESULTS: In the first 3 months, considerable time was expended in both groups, with the time spent by physicians a little (but significantly) longer for the insulin group. The total time expended by all care providers was approximately 1.3-fold greater for the insulin compared with the non-insulin group. The total cost and total cost/min for the insulin group was almost twice that for the non-insulin group. Over the 5-month period, mean HbA1c in the non-insulin group improved from 8.0% to 6.5%, with 72% achieving a glycemic target of HbA1c ≤ 6.5%. In contrast, in the insulin group, mean HbA1c improved from 9.4% to 7.6%, with only 39% achieving the target. There were no reports of major hypoglycemic events in either group and body mass index remained stable. CONCLUSIONS: The insulin therapy for T2DM can be achieved safely and effectively at outpatient clinics, even though it requires considerably more time and resources than non-insulin therapy.

Magnesium excretion in C. elegans requires the activity of the GTL-2 TRPM channel.

Systemic magnesium homeostasis in mammals is primarily governed by the activities of the TRPM6 and TRPM7 cation channels, which mediate both uptake by the intestinal epithelial cells and reabsorption by the distal convoluted tubule cells in the kidney. In the nematode, C. elegans, intestinal magnesium uptake is dependent on the activities of the TRPM channel proteins, GON-2 and GTL-1. In this paper we provide evidence that another member of the TRPM protein family, GTL-2, acts within the C. elegans excretory cell to mediate the excretion of excess magnesium. Thus, the activity of GTL-2 balances the activities of the paralogous TRPM channel proteins, GON-2 and GTL-1.

Modulation of EGF receptor-mediated vulva development by the heterotrimeric G-protein Galphaq and excitable cells in C. elegans.

The extent to which excitable cells and behavior modulate animal development has not been examined in detail. Here, we demonstrate the existence of a novel pathway for promoting vulval fates in C. elegans that involves activation of the heterotrimeric Galphaq protein, EGL-30. EGL-30 acts with muscle-expressed EGL-19 L-type voltage-gated calcium channels to promote vulva development, and acts downstream or parallel to LET-60 (RAS). This pathway is not essential for vulval induction on standard Petri plates, but can be stimulated by expression of activated EGL-30 in neurons, or by an EGL-30-dependent change in behavior that occurs in a liquid environment. Our results indicate that excitable cells and animal behavior can provide modulatory inputs into the effects of growth factor signaling on cell fates, and suggest that communication between these cell populations is important for normal development to occur under certain environmental conditions.