TRPM channels are required for rhythmicity in the ultradian defecation rhythm of C. elegans
- Claire SM Kwan†1,
- Rafael P Vázquez-Manrique†1,
- Sung Ly1,
- Kshamata Goyal1 and
- Howard A Baylis1Email author
© Kwan et al; licensee BioMed Central Ltd. 2008
Received: 21 February 2008
Accepted: 21 May 2008
Published: 21 May 2008
Ultradian rhythms, rhythms with a period of less than 24 hours, are a widespread and fundamental aspect of life. The mechanisms underlying the control of such rhythms remain only partially understood. Defecation in C. elegans is a very tightly controlled rhythmic process. Underlying the defecation motor programme is an oscillator which functions in the intestinal cells of the animal. This mechanism includes periodic calcium release and subsequent intercellular calcium waves which in turn regulate the muscle contractions that make up the defecation motor programme. Here we investigate the role of TRPM cation channels in this process.
We use RNA interference (RNAi) to perturb TRPM channel gene expression. We show that combined knock down of two of the TRPM encoding genes, gon-2 and gtl-1, results in an increase in the variability of the cycle but no change in the mean, in normal culture conditions. By altering the mean using environmental (temperature) and genetic approaches we show that this increase in variability is separable from changes in the mean. We show that gon-2 and gtl-1 interact with components of the calcium signalling machinery (itr-1 the C. elegans inositol 1,4,5-trisphosphate receptor) and with plasma membrane ion channels (flr-1 and kqt-3) which are known to regulate the defecation oscillator. Interactions with these genes result in changes to the mean period and variability. We also show that knocking down a putative transcription factor can suppress the increased variability caused by reduction of gon-2 and gtl-1 function. We also identify a previously unrecognised tendency of the defecation cycle to compensate for cycles with aberrant length by adjusting the length of the following cycle.
Thus TRPM channels regulate the variability of the defecation oscillator in C. elegans. We conclude that the mean and the variability of the defecation oscillator are separable. Our results support the notion that there is a strong underlying pacemaker which is able to function independently of the observable defecation rhythm and is not perturbed by increases in the variability of the cycle.
The interaction of gon-2 and gtl-1 with other components of the oscillator shows that TRPM channels play an important role in the oscillator machinery. Such a role may be through either regulation of cation levels or membrane properties or both. Specifically our results support previous proposals that gon-2 and gtl-1 regulate IP3 signalling and that kqt-3 may act by altering calcium influx.
Our results provide novel insights into the properties of the defecation oscillator and thus to our understanding of ultradian rhythms.
In this work we focus on the role TRPM (Transient Receptor Potential Melastatin) channels [11, 12]. TRPM channels are a subgroup of the TRP channel superfamily [11, 12]. Mammalian genomes encode eight members of the TRPM family, which can be divided into two groups consisting of TRPM1,3,6 and 7 and TRPM2,4,5 and 8. TRPM channels have a range of biophysical properties exhibiting both selective and non-selective cation permeation. TRPM6 and 7 have been implicated in magnesium homeostasis in the intestine and kidney [13–18] and mutations in TRPM6 underlie inherited defects in magnesium and calcium uptake [13, 16]. C. elegans has three members of the TRPM channel family, gtl-1, gtl-2 and gon-2. gon-2 is known to be involved in the regulation of mitosis during gonadogenesis [19–23]. gon-2;gtl-1 double mutants have a severe growth defect which is ameliorated by the presence of high (typically 40 mM added Mg2+) in the growth medium . Furthermore it has been shown that gtl-1 double mutants have reduced Mg2+ levels in the intestine. Thus it has been suggested that these two genes have a key role in the regulation of the uptake of magnesium and other cations in the intestine .
Recent work has shed light on the function of gon-2 and gtl-1. Teramoto et al. suggested, based on genetic, functional and electrophysiological data, that gon-2 and gtl-1 function as separate channels with gon-2 contributing to a current with similarities to the previously characterised IORCa . More recent analysis suggests that gon-2 and gtl-1 are both required for IORCa activity, that the channel is calcium selective and that channels encoded singly by gon-2 or gtl-1 have very similar properties. There are several other differences between the results from these two groups the causes of which remain unclear (see discussion in ).
Teramoto et al.  also showed that gtl-1 and gon-2;gtl-1 double mutants have a disrupted defecation cycle under certain conditions . However successful culture of gon-2;gtl-1 double mutants requires that they are grown in unusually high Mg2+ (typically 40 mM added Mg2+) compared to normal C. elegans culture conditions of 1 mM added Mg2+. As a result, experiments are performed either on worms in 40 mM Mg2+ or on worms which have been shifted from 40 mM Mg2+ to other conditions, thus exposing worms to either abnormally high levels of magnesium or large rapid changes in cation level. This may result in compounding various effects on the defecation cycle. Recently Xing et al., have also shown that gon-2 and gtl-1 mutants disrupt the defecation cycle .
In this work we show, using RNA mediated interference (RNAi), that in normal Mg2+ conditions, ablation of gon-2 and gtl-1 substantially increases the variability of the defecation rhythm without significantly altering the period of the cycle. Therefore, we hypothesise that these genes function to control the variability (i.e. the rhythm) of the cycle. We show by analysing the behaviour of defective gon-2(RNAi);gtl-1(RNAi) worms at different temperatures and in clk mutants that this increase in variability is independent of changes in the period. Further we show that this increase in variability can be suppressed under certain genetic condition. Thus we propose that period and variability can be independently regulated. We also show that gtl-1 and gon-2 interact with the itr-1, flr-1 and kqt-3 genes, which also affect the variation and the rate of the defecation oscillator.
The TRPM channels gon-2 and gtl-1 are required for a rhythmic defecation cycle
We set out to understand the nature of the input of TRPM channels into the defecation oscillator. To circumvent the problems associated with culturing worms at high magnesium (see introduction) and further define the role of TRPM channels in the defecation rhythm we used RNAi. Knock down by RNAi does not require that the worms are fertile (gon-2 null animals are sterile) and RNAi animals grow sufficiently well in normal culture conditions for analysis (CSMK unpublished).
In gon-2(RNAi);gtl-1(RNAi) knock-down worms, the mean period of the cycle remains unaltered whilst the variability of the cycle is substantially increased (Figure 2A). Thus the mean and the mean C.V. for the control animals are 46.4 s and 5.5%, whereas those for gon-2(RNAi);gtl-1(RNAi) animals are 46.5 s and 21.1%. That is, the variability has increased by nearly four fold. Examples of the pattern of defecation periods in individual worms (figure 2B) show that individual worms have increased variability, excluding the possibility that this effect results from variable levels of RNAi. This strongly suggests that gon-2 and gtl-1 function together in regulating the rhythmicity of the cycle.
The arrhythmicity conferred by gon-2(RNAi);gtl-1(RNAi) knock-down is independent of the mean of the rhythm
Our results suggest that the increase in variability caused by knocking down gon-2;gtl-1 function is largely independent of the mean. To test whether mean period and variability could be manipulated independently we perturbed the mean of the defecation oscillator using environmental and genetic treatments.
A wide range of mutants have been isolated which alter the period of the defecation oscillator (see  for review). clk mutants have changes in a range of rates and rhythms including life span and defecation. In clk mutants the defecation period is extended [27–29]. clk-1 encodes a putative hydroxylase required for ubiquinone biosynthesis  whilst the molecular nature of clk-3 is unknown. In our conditions, the period of defecation in both clk-1 and clk-3 mutants is increased by about 40%. We therefore knocked down gon-2 and gtl-1 by RNAi in clk-1(qm30) and clk-3(qm38) animals (Figure 3B). In both cases the period of defecation remained unaltered following RNAi, e.g. in the clk-1 animals it was 66 s whilst in clk-1(qm30);gon-2(RNAi);gtl-1(RNAi) animals it was 63 s. However in both cases RNAi of gon-2 and gtl-1 substantially increased the variability (see figure 3). In the case of clk-1 the C.V. was increased from 7 to 23% and in clk-3 from 4 to 18%. These experiments confirm that the period and rhythmicity of the process can be manipulated independently.
TRPM channel knock-down interacts with other components of the defecation oscillator
We screened ten other genes which are known to be involved in the defecation programme for interactions with gon-2(RNAi);gtl-1(RNAi). We detected strong interaction in four cases discussed below and no interaction with crt-1(jh101), fat-3(wa22), kqt-2(ok732), lin-42(RNAi), unc-43(sa200) and unc-75(e950).
gon-2 and gtl-1 encode putative plasma membrane cation channels [21, 23]. We therefore tested whether they interact with other genes that are known to regulate defecation and to regulate plasma membrane ion flux. flr-1 encodes a member of DEG/ENaC sodium channel family . flr-1 animals have a very fast defecation cycle  (mean 18 s in our conditions) (Figure 4B). They also have a very large variability (C.V = 19%). When we knocked down gon-2 and gtl-1 in these worms the variability showed a further very large increase (Figure 4B). In addition the mean cycle length was increased to close to the wild-type. Thus gon-2(RNAi);gtl-1(RNAi) suppresses the short defecation cycle phenotype of flr-1 animals and enhances the variability of flr-1 mutants. kqt-3 encodes a member of the KCNQ family of potassium channels  which is known to play a role in defecation . kqt-3 (lf) animals have an increased mean and high variability. Reduction of gon-2 and gtl-1 in these animals causes a suppression of the increased mean with little or no effect on the variability (Figure 4B)
RNAi of the zinc finger protein R08E3.4 suppresses the increased variability caused by reduction in gon-2 and gtl-1 function
We identified a further gene that modified the effect of reducing gon-2 and gtl-1 function Using RNAi we tested the effect of knocking down lin-42 (the C. elegans per homologue ) and a gene R08E3.4, which is also implicated in heterochronic regulation (Gardner and Rougvie pers. comm.). R08E3.4 is a zinc finger protein with similarities to the Ikaros family of transcription factors. lin-42 had little effect but, strikingly, R08E3.4 RNAi was able to substantially reduce the variability which results from gon-2(RNAi);gtl-1(RNAi) returning the variability to near wild-type levels (Figure 4C). The animals in these experiments showed the phenotypic effects (reduced fertility and growth) typical of gon-2(RNAi);gtl-1(RNAi) ruling out the possibility that the reduction in variability was due to reduced levels of RNAi. Once again the mean of the period was unaffected. Thus the variability that results from gon-2(RNAi);gtl-1(RNAi) can be suppressed by other genes.
Analysis of gon-2(RNAi);gtl-1(RNAi) animals reveals self-regulation in the defecation oscillator
The results presented in this paper demonstrate that disruption of the TRPM channels gon-2 and gtl-1 results in an increased variability in the defecation oscillator. By altering the mean, using mutations (in clk-1 and clk-3) and temperature, in gon-2;gtl-1 depleted animals we show that the variability of the oscillator and its mean can be manipulated independently. By testing interactions with other genes we identify informative interactions between gon-2;gtl-1 and itr-1, flr-1 and kqt-3 all genes known to be involved in defecation.
By using RNAi of gon-2 and gtl-1 we have been able to clearly define the major effect of knocking down these channels, in normal culture conditions. It is clear that reducing the activity of gon-2 and gtl-1 together results in a substantial increase in the variability of the rhythm without significantly affecting the mean. A similar pattern of disruption is seen in inx-16 mutants . Importantly this increase in variability is independent of alterations in the mean caused by either genetic (clk-1 and clk-3) or environmental (temperature) manipulation. This suggests that the underlying oscillator is still oscillating in these mutants and that the mechanisms that set the period are also largely unaffected. It also suggests that gon-2 and gtl-1 are part of a mechanism that maintains regularity and therefore presumably the stability of the oscillator. It should be noted however that experiments using mutants of gon-2 and gtl-1 show that in these animals the period of the cycle is also altered [24, 26]. This difference may reflect more severe impairment of channel function in mutant animals. We also found that in certain backgrounds, in particular those with disrupted membrane ion flux, gon-2(RNA)i;gtl-1(RNAi) also alters the mean period. We suggest that gon-2 and gtl-1 are part of a mechanism which is required for the stability of the oscillator in normal circumstances.
One possible explanation for the variability observed in the gon-2(RNAi);gtl-1(RNAi) animals stems from the suggestion that the period of the oscillator actually reflects an underlying faster pacemaker with a period of about 15 seconds, with suppression of intermediate calcium signalling events . Thus in gon-2;gtl-1 animals it could be that they execute the DMP on random underlying pacemaker events. We did not detect such a trend; most of the variability appears to result from continuous spread about the mean. However we cannot rule out that some may reflect a changed response to the pacemaker as identifying this would require very large sample sizes.
How can we explain the observation that we can disrupt the rhythmicity of the cycle without disrupting the mean? One explanation for the ability of the animals to maintain a normal period but have increased variability is that there is a mechanism that sets the period and that the oscillator fluctuates around this input, so that disruption of gon-2 and gtl-1 compromises the ability of the oscillator to adhere to the input period. This is clearly the case to some extent as genes such as clk-1 change the mean of the cycle without destabilising the oscillator. Alternatively the oscillator may be self regulating and so able to maintain a normal period even when its stability is disrupted. Our analysis of the cycle suggests that this may also be the case. We observed that there is strong tendency for increases or decreases in the cycle length to be followed by inverse changes on the next cycle. We also observed that there is some correlation in the magnitude of such changes. This property could reflect a passive property of the system or could reflect an active mechanism to compensate for divergent cycles. Such a mechanism may exist to maintain the correct flow of material through the intestine. In normal worms the cycle is very tightly regulated, suggesting that the period of the cycle is important to the animal. That this is still the case in gon-2(RNAi);gtl-1(RNAi) animals again points to the machinery setting the period being largely intact.
In our experiments we detect a clear synergy between gon-2, gtl-1 and itr-1. itr-1 has been shown to be downstream of plc-3 in the defecation oscillator  and analysis of interactions between gon-2 and gtl-1 and plc-3 has strongly suggested a link between the TRPM channels and IP3 signalling . Our results further support this model. One possible explanation for the synergy of gon-2;gtl-1 and itr-1 is that in gon-2;gtl-1 mutants calcium uptake is disrupted either directly or indirectly which results in depleted stores resulting in, either misregulation of itr-1 or altered levels of calcium release from the stores. Interestingly, Nehrke et al  observed that partial depletion of sca-1, the worm SERCA homologue, did not result in arrhythmia. This suggests that proper store filling is not a key determinant of rhythmicity. Thus this role for TRPM channels may be unlikely. Another possible explanation for the synergistic effect with ITR-1 is that Ca2+ flux through GON-2 and GTL-1 maybe acting on ITR-1 directly to modulate its activity (see  for an extensive discussion of this idea) as IP3Rs are known to be regulated by calcium levels . Finally Ca2+ influx through GON-2 and GTL-1 may contribute directly to calcium waves induced by ITR-1.
The effect of depleting flr-1 or kqt-3 together with gon-2 and gtl-1 suggests that the functions of gon-2 and gtl-1 are linked to the properties of the plasma membrane. flr-1 mutants have altered means and rhythmicity. Simultaneous knock-down of gon-2;gtl-1in flr-1 mutants results in shifts in both the mean and the C.V. This suggests that either, flr-1 and gon-2;gtl-1 act in parallel to maintain rhythmicity or possibly that one acts on residual activity in the other to further disrupt its function. flr-1 encodes a putative plasma membrane DEG/ENaC type channel in the intestine and gon-2;gtl-1 are also likely to be plasma membrane cation channels. One possibility is that the interaction between these two channel types is mediated by alterations to the electrophysiological properties of the membrane. Other membrane components also alter defecation, for example kqt-2 and kqt-3 which are both members of the KCNQ M-type K+ channels. We show that simultaneous depletion of gon-2, gtl-1 and kqt-3 results in suppression of the increased mean resulting from kqt-3 depletion. Thus gon-2 and gtl-1 maybe targets of kqt-3 action. Nehrke et al.  proposed that kqt channels exert their effect by regulating calcium influx. If gon-2 and gtl-1 act by contributing to IORCa and thus calcium influx , then our results may support this model. We speculate that flr-1 also alters this process. However it is unclear why modulation of gon-2 and gtl-1 activity by these mechanisms would produce a change in the mean period when this does not occur on depletion in wild-type animals. It is clear that the interactions of these components require further analysis.
Finally we have identified a putative transcription factor, R08E3.4, depletion of which suppresses the gon-2(RNAi);gtl-1(RNAi) phenotype. The mechanism of this action is unclear. Further analysis of this interaction should provide new insights into the function of gon-2 and gtl-1 in defecation.
The defecation cycle of C. elegans provides an example of an oscillatory mechanism that controls an ultradian rhythm. Using RNAi we show that two orthologs of the TRPM cation channel family are important component of the part of the mechanism which ensures regularity. We show that the oscillator is self adjusting with respect to period and that this ability is not disrupted in gon-2 and gtl-1 depleted animals. These results indicate that there is a strong underlying pacemaker which is able to function independently of the observable defecation rhythm and is not perturbed by disruption of the variability of the cycle.
We also show that gon-2 and gtl-1 interact with both calcium signalling molecules (itr-1) and other channels in the plasma membrane (flr-1, kqt-3). Thus the TRPM channels play an important role in the machinery of the oscillator. Our results are compatible with a model in which gon-2 and gtl-1 regulate IP3 signalling and in which kqt-3 (and possible flr-1) regulate gon-2 and gtl-1 function.
Our results provide a novel insight into the properties of the defecation oscillator and thus to our understanding of ultradian rhythms.
Worm husbandry and strains
C. elegans was maintained and cultured using standard conditions . All worms were grown on NGM plates; containing 1 mM added MgCl2 and 1 mM added CaCl2 unless otherwise stated. We used the following strains: N2 (Bristol strain, wild type), MQ130: clk-1(qm30) , MQ131: clk-3(qm38) [37, 38], JC55: flr-1(ut11) , JT73: itr-1(sa73) (a gift of J. Thomas) , TM542: kqt-3(tm542) (a gift of S. Mitani) and HB201: gtl-1(ok375). HB201 is a derivative of VC244 out-crossed to N2 three times.
RNA mediated interference
Double stranded RNA for RNAi was introduced by injection. Single stranded RNA for each strand was produced by in vitro transcription (MegaScript, Ambion) from clones containing approximately 1 kb of each cDNA amplified by PCR and cloned into pGEM-T (Promega). Details of clones are available on request. As a control dsRNA from the cat (chloramphenicol acetyltransferase) gene of E. coli was used. This was produced from the vector pPD136.60 (a gift of A. Fire). Preparation and injection of dsRNA was as described in . Adult animals were injected, F1 progeny were then analyzed.
Measurement and analysis of the defecation rhythm
Animals were grown, for at least two generations at the test temperature, normally 20°C. Animals were routinely cultured and measured on E. coli OP50. Analysis was performed in controlled temperature rooms. The length of the DMP was scored as the time between two posterior body wall contractions (pBoc) [See Figure 1]. To establish the parameters of the defecation rhythm we used the approach of Kippert (pers comm.). L4 worms were each scored for 15 consecutive cycles (unless otherwise noted) for each experiment. In most experiments more than 25 worms were scored and experiments were performed in duplicate or triplicate. Results from representative experiments are shown. Where appropriate the behaviour of untreated animals of a strain was confirmed as being similar to that previously reported.
For each data set we calculated the mean period of the defecation cycle and SEM. To measure the variation of the cycle we calculated the mean coefficient of variation which is the mean of the C.V.s for each worm in the experiment. The significance of differences in values was determined using unpaired t-tests.
Statistical analysis of the defecation oscillator's behaviour
We defined the period of a cycle (n) as T(n). We defined an increase (represented by +) in cycle length as T(n+1)- T(n) > 1 sec. A decrease (represented by -) was defined as T(n+1)- T(n) < -1 sec. Values of ≥ -1 or ≤ 1 were defined as no change (0). In designing these criteria we assumed that a resolution of less than 1 sec in the difference was beyond the accuracy inherent in the experimental procedure. We defined nine possible combinations of the change in period between two cycles: 00, 0-, 0+, --, -0, -+, ++, +0, +-. To analyse a data set each pair of cycle differences was assigned to a particular class. We ignored members of the 00, 0+ and 0- classes as they are uninformative for this analysis. The numbers of members of the other classes were pooled as; "0-" & "0+", "++" & "--", "+-" & "-+". Results for a representative data set are shown. The original set contained 15 cycles for 25 worms yielding 325 cycle difference pairs.
To assess the correlation in magnitude between cycles we used the same data set. We determined the values of T(n+1)-T(n) for all pairs of period times. We censured all pairs in which either member of the pair had a magnitude of -1, 0 or 1. We then plotted T(n+1)- T(n) against T(n+2)-T(n+1). To assess correlation we used a Pearson test and we used linear regression (using GraphPad Prism) to derive the slope of the line. Significance was calculated using a two tailed T-test.
We are grateful to A. Rougvie and H. Gardner for sharing information on R08E3.4. We are grateful to Dr F. Kippert for advice and sharing reagents. We are grateful to A. Fire and J. Thomas for gifts of materials used in this research. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR) and by the National Bioresource Project for the Experimental Animal C. elegans, Japan. We thank D. Walker for advice and helpful discussion and C. Ragnauth for assistance with early stages of this work. This work was supported by the BBSRC (NSL, KG, RVM) and MRC (CSMK and HAB).
- Liu DW, Thomas JH: Regulation of a periodic motor program in C. elegans. J Neurosci. 1994, 14 (4): 1953-1962.PubMedGoogle Scholar
- Baylis HA: VAV's got rhythm. Cell. 2005, 123 (1): 5-7. 10.1016/j.cell.2005.09.018.View ArticlePubMedGoogle Scholar
- Thomas JH: Genetic analysis of defecation in Caenorhabditis elegans. Genetics. 1990, 124 (4): 855-872.PubMed CentralPubMedGoogle Scholar
- Branicky R, Hekimi S: What keeps C. elegans regular: the genetics of defecation. Trends Genet. 2006, 22 (10): 571-579. 10.1016/j.tig.2006.08.006.View ArticlePubMedGoogle Scholar
- Iwasaki K, Liu DW, Thomas JH: Genes that control a temperature-compensated ultradian clock in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1995, 92 (22): 10317-10321. 10.1073/pnas.92.22.10317.PubMed CentralView ArticlePubMedGoogle Scholar
- Dal Santo P, Logan MA, Chisholm AD, Jorgensen EM: The inositol trisphosphate receptor regulates a 50-second behavioral rhythm in C. elegans. Cell. 1999, 98 (6): 757-767. 10.1016/S0092-8674(00)81510-X.View ArticlePubMedGoogle Scholar
- Espelt MV, Estevez AY, Yin X, Strange K: Oscillatory Ca2+ signaling in the isolated Caenorhabditis elegans intestine: role of the inositol-1,4,5-trisphosphate receptor and phospholipases C b and g. J Gen Physiol. 2005, 126 (4): 379-392. 10.1085/jgp.200509355.PubMed CentralView ArticlePubMedGoogle Scholar
- Nehrke K, Denton J, Mowrey W: Intestinal Ca2+ wave dynamics in freely moving C. elegans coordinate execution of a rhythmic motor program. Am J Physiol Cell Physiol. 2008, 294 (1): C333-44. 10.1152/ajpcell.00303.2007.View ArticlePubMedGoogle Scholar
- Peters MA, Teramoto T, White JQ, Iwasaki K, Jorgensen EM: A calcium wave mediated by gap junctions coordinates a rhythmic behavior in C. elegans. Curr Biol. 2007, 17 (18): 1601-1608. 10.1016/j.cub.2007.08.031.View ArticlePubMedGoogle Scholar
- Teramoto T, Iwasaki K: Intestinal calcium waves coordinate a behavioral motor program in C. elegans. Cell Calcium. 2006, 40 (3): 319-327. 10.1016/j.ceca.2006.04.009.View ArticlePubMedGoogle Scholar
- Venkatachalam K, Montell C: TRP channels. Annu Rev Biochem. 2007, 76: 387-417. 10.1146/annurev.biochem.75.103004.142819.PubMed CentralView ArticlePubMedGoogle Scholar
- Pedersen SF, Owsianik G, Nilius B: TRP channels: an overview. Cell Calcium. 2005, 38 (3-4): 233-252. 10.1016/j.ceca.2005.06.028.View ArticlePubMedGoogle Scholar
- Walder RY, Landau D, Meyer P, Shalev H, Tsolia M, Borochowitz Z, Boettger MB, Beck GE, Englehardt RK, Carmi R, Sheffield VC: Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat Genet. 2002, 31 (2): 171-174. 10.1038/ng901.View ArticlePubMedGoogle Scholar
- Hoenderop JG, Bindels RJ: Epithelial Ca2+ and Mg2+ channels in health and disease. J Am Soc Nephrol. 2005, 16 (1): 15-26. 10.1681/ASN.2004070523.View ArticlePubMedGoogle Scholar
- Schlingmann KP, Waldegger S, Konrad M, Chubanov V, Gudermann T: TRPM6 and TRPM7--Gatekeepers of human magnesium metabolism. Biochim Biophys Acta. 2007, 1772 (8): 813-821.View ArticlePubMedGoogle Scholar
- Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, Vitzthum H, Klingel K, Kratz M, Haddad E, Ristoff E, Dinour D, Syrrou M, Nielsen S, Sassen M, Waldegger S, Seyberth HW, Konrad M: Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet. 2002, 31 (2): 166-170. 10.1038/ng889.View ArticlePubMedGoogle Scholar
- Schmitz C, Perraud AL, Johnson CO, Inabe K, Smith MK, Penner R, Kurosaki T, Fleig A, Scharenberg AM: Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. Cell. 2003, 114 (2): 191-200. 10.1016/S0092-8674(03)00556-7.View ArticlePubMedGoogle Scholar
- Voets T, Nilius B, Hoefs S, van der Kemp AW, Droogmans G, Bindels RJ, Hoenderop JG: TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption. J Biol Chem. 2004, 279 (1): 19-25. 10.1074/jbc.M311201200.View ArticlePubMedGoogle Scholar
- Harteneck C, Plant TD, Schultz G: From worm to man: three subfamilies of TRP channels. Trends Neurosci. 2000, 23 (4): 159-166. 10.1016/S0166-2236(99)01532-5.View ArticlePubMedGoogle Scholar
- Kahn-Kirby AH, Bargmann CI: TRP channels in C. elegans. Annu Rev Physiol. 2006, 68: 719-736. 10.1146/annurev.physiol.68.040204.100715.View ArticlePubMedGoogle Scholar
- Baylis HA, Goyal K: TRPM channel function in Caenorhabditis elegans. Biochem Soc Trans. 2007, 35 (Pt 1): 129-132.View ArticlePubMedGoogle Scholar
- Sun AY, Lambie EJ: gon-2, a gene required for gonadogenesis in Caenorhabditis elegans. Genetics. 1997, 147 (3): 1077-1089.PubMed CentralPubMedGoogle Scholar
- West RJ, Sun AY, Church DL, Lambie EJ: The C. elegans gon-2 gene encodes a putative TRP cation channel protein required for mitotic cell cycle progression. Gene. 2001, 266 (1-2): 103-110. 10.1016/S0378-1119(01)00373-0.View ArticlePubMedGoogle Scholar
- Teramoto T, Lambie EJ, Iwasaki K: Differential regulation of TRPM channels governs electrolyte homeostasis in the C. elegans intestine. Cell Metab. 2005, 1 (5): 343-354. 10.1016/j.cmet.2005.04.007.PubMed CentralView ArticlePubMedGoogle Scholar
- Estevez AY, Roberts RK, Strange K: Identification of store-independent and store-operated Ca2+ conductances in Caenorhabditis elegans intestinal epithelial cells. J Gen Physiol. 2003, 122 (2): 207-223. 10.1085/jgp.200308804.PubMed CentralView ArticlePubMedGoogle Scholar
- Xing J, Yan X, Estevez A, Strange K: Highly Ca2+-selective TRPM channels regulate IP3-dependent oscillatory Ca2+ signaling in the C. elegans intestine. J Gen Physiol. 2008, 131 (3): 245-255. 10.1085/jgp.200709914.PubMed CentralView ArticlePubMedGoogle Scholar
- Lakowski B, Hekimi S: Determination of life-span in Caenorhabditis elegans by four clock genes. Science. 1996, 272 (5264): 1010-1013. 10.1126/science.272.5264.1010.View ArticlePubMedGoogle Scholar
- Branicky R, Shibata Y, Feng J, Hekimi S: Phenotypic and suppressor analysis of defecation in clk-1 mutants reveals that reaction to changes in temperature is an active process in Caenorhabditis elegans. Genetics. 2001, 159 (3): 997-1006.PubMed CentralPubMedGoogle Scholar
- Wong A, Boutis P, Hekimi S: Mutations in the clk-1 gene of Caenorhabditis elegans affect developmental and behavioral timing. Genetics. 1995, 139 (3): 1247-1259.PubMed CentralPubMedGoogle Scholar
- Ewbank JJ, Barnes TM, Lakowski B, Lussier M, Bussey H, Hekimi S: Structural and functional conservation of the Caenorhabditis elegans timing gene clk-1. Science. 1997, 275 (5302): 980-983. 10.1126/science.275.5302.980.View ArticlePubMedGoogle Scholar
- Walker DS, Gower NJ, Ly S, Bradley GL, Baylis HA: Regulated disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis elegans reveals new functions in feeding and embryogenesis. Mol Biol Cell. 2002, 13 (4): 1329-1337. 10.1091/mbc.01-08-0422.PubMed CentralView ArticlePubMedGoogle Scholar
- Take-Uchi M, Kawakami M, Ishihara T, Amano T, Kondo K, Katsura I: An ion channel of the degenerin/epithelial sodium channel superfamily controls the defecation rhythm in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1998, 95 (20): 11775-11780. 10.1073/pnas.95.20.11775.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei AD, Butler A, Salkoff L: KCNQ-like potassium channels in Caenorhabditis elegans. Conserved properties and modulation. J Biol Chem. 2005, 280 (22): 21337-21345. 10.1074/jbc.M502734200.View ArticlePubMedGoogle Scholar
- Jeon M, Gardner HF, Miller EA, Deshler J, Rougvie AE: Similarity of the C. elegans developmental timing protein LIN-42 to circadian rhythm proteins. Science. 1999, 286 (5442): 1141-1146. 10.1126/science.286.5442.1141.View ArticlePubMedGoogle Scholar
- Foskett JK, White C, Cheung KH, Mak DO: Inositol trisphosphate receptor Ca2+ release channels. Physiol Rev. 2007, 87 (2): 593-658. 10.1152/physrev.00035.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Lewis JA, Fleming JT: Basic culture methods. Methods Cell Biol. 1995, 48: 3-29.View ArticlePubMedGoogle Scholar
- Hekimi S, Boutis P, Lakowski B: Viable maternal-effect mutations that affect the development of the nematode Caenorhabditis elegans. Genetics. 1995, 141 (4): 1351-1364.PubMed CentralPubMedGoogle Scholar
- Katsura I, Kondo K, Amano T, Ishihara T, Kawakami M: Isolation, characterization and epistasis of fluoride-resistant mutants of Caenorhabditis elegans. Genetics. 1994, 136 (1): 145-154.PubMed CentralPubMedGoogle Scholar
- Timmons L: C elegans: Delivery Methods for RNA Interference in C. elegans. Methods and Applications. Edited by: Strange K. 2006, Clifton, NJ , Humana Press, 351: 119-126.View ArticleGoogle Scholar
- Avery L, Thomas JH: Feeding and Defecation. C elegans II. Edited by: Riddle DLBTMBJPJR. 1997, Cold Spring Harbor , Cold Spring Harbor Press, 679-716.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.