Elsevier

Brain Research

Volume 821, Issue 1, 6 March 1999, Pages 160-168
Brain Research

Research report
Functional reconstitution of a heteromeric cyclic nucleotide-gated channel of Caenorhabditis elegans in cultured cells

https://doi.org/10.1016/S0006-8993(99)01111-7Get rights and content

Abstract

The tax-4 and tax-2 genes of Caenorhabditis elegans are essential for normal olfaction, gustation, and thermosensation, suggesting that they have a role in sensory transduction. The predicted products of these genes are similar to the cyclic nucleotide-gated (CNG) channel subunits used in vertebrate vision and olfaction: TAX-4 is highly related to those α subunits, while TAX-2 is most closely related to the β subunits of the rod phototransduction channels. TAX-4 has previously been shown to form a highly sensitive cGMP-gated channel when expressed in human HEK293 cells. Here we show that TAX-4 and TAX-2 form a heteromeric channel when expressed in HEK293 cells, but TAX-2 does not form a channel on its own. Since these genes are expressed in the same neurons, most of the native channels in C. elegans are likely to be hetero-oligomers of TAX-4 and TAX-2 subunits, with TAX-4 as the α subunit and TAX-2 acting as a modifying β subunit. The heteromeric TAX-4/TAX-2 channel is 25-fold less sensitive to cGMP than the TAX-4 channel, but it remains highly selective for cGMP over cAMP. The heteromeric channel and the TAX-4 homomeric channel differ in their blockage by divalent cations and in their single channel properties. These results suggest that cGMP is used as the second messenger during sensory signal transduction in C. elegans, and that distinct roles for α and β subunits of CNG channels are conserved in vertebrate and invertebrate signal transduction.

Introduction

Vertebrate cyclic nucleotide-gated (CNG) nonselective cation channels play a central role in visual and olfactory signal transduction. These channels are gated by cAMP or cGMP to allow Na+ and Ca2+ entry and depolarization, and they are distantly related to the voltage-gated channel such as the Shaker potassium channel. Sensory signal transduction in vertebrate visual and olfactory systems is initiated by G protein-coupled receptors. In photoreceptor cells, light-activated rhodopsin activates a phosphodiesterase, which decreases the concentration of cGMP and closes the channel, an event leading to hyperpolarization of the photoreceptor. In olfactory neurons, odorant receptors activate an adenylate cyclase, which increases cAMP and opens the channel in the cilia and dendrites. Channel opening leads to depolarization of the olfactory neuron and production of an action potential 13, 30.

The native rod photoreceptor and olfactory CNG channels are believed to be composed of two distinct subunits (α and β, or 1 and 2). The α subunit by itself can form functional CNG channels when expressed in heterologous systems. The β subunit does not function as a CNG channel by itself, but it forms a heteromeric channel whose properties more closely resemble those of the native channel when co-expressed with the corresponding α subunit 2, 3, 10, 18, 19. More complex channels may also exist: the native vertebrate olfactory CNG channel may consist of a heteromer with three different subunits [27].

Interestingly, recent studies have suggested that the CNG channel may also have a role in sensory development. The olfactory CNG channel is activated directly by nitric oxide (NO), and NO synthase is expressed transiently in the olfactory epithelium during development and neuronal regeneration 4, 26. Furthermore, the β subunit alone can be activated by NO to form Ca2+ channels [5]. This raised the possibility that the CNG channel may be activated during sensory development even without an external olfactory stimulus.

Behavioral, genetic and cellular analyses revealed that two CNG channel homologs, TAX-4 and TAX-2, play important roles in thermosensation and chemosensation in the nematode Caenorhabditis elegans7, 17. TAX-4 and TAX-2 display highest sequence similarities with the α and β subunits of the retinal rod photoreceptor channel, respectively. The phenotypes of tax-4 and tax-2 mutants and expression patterns of tax-4 and tax-2 genes are nearly identical. Both mutants exhibit almost random movement on an unseeded plate with a radial temperature gradient (athermotactic phenotype) [23]. They fail to respond to salts and AWC-sensed odorants (benzaldehyde, 2-butanone, and isoamyl alcohol), and are slightly abnormal in dauer larva formation. The tax-4 and tax-2 genes are expressed in sensory neurons including AFD thermosensory neurons, ASE gustatory neurons detecting salts, AWC olfactory neurons, and ASI and ASJ neurons mediating the dauer formation, all of which are affected by tax-4 or tax-2 mutations. Besides their sensory defects, both mutants show axonal outgrowth defects in some sensory neurons, suggesting that the TAX-4/TAX-2 proteins are also required for neuronal development 7, 8. The extensive similarity of their functions suggests that TAX-2 and TAX-4 might form a heteromeric channel, but other models are also possible. We wished to examine the properties of the TAX-2/4 channels. It would be very difficult to get the native channels from C. elegans, and such examination is practical only by the expression in heterologous, cultured cells. As the first step, we expressed the TAX-4 protein in HEK293 cells and showed that TAX-4 alone could form CNG channels and that these channels were highly sensitive to cGMP [17].

Here we show that TAX-4 and TAX-2 function as α and β subunits of the CNG channel in HEK293 cells, respectively, and that the heteromeric channel is much more sensitive to cGMP than to cAMP. Our results support the notion that cGMP serves as a second messenger in C. elegans sensory signal transduction. We also describe unique properties of the C. elegans CNG channels.

Section snippets

Molecular biology

Full-length tax-2 cDNAs were generated by fusing a partial tax-2 cDNA [7]with cDNA fragments generated from wild-type animal by RT-PCR. All the RT-PCR products were sequenced and found to be wild-type. The complete tax-4 and tax-2 cDNAs were inserted into the polylinker site of pCI (Promega Madison, WI) [17]. Human embryonic kidney (HEK) 293 cells were grown on poly-d-lysine-coated coverslips in DMEM with 10% FCS and 10% CO2. For expression of a single subunit alone, cells were transfected with

Cyclic nucleotide sensitivity of TAX-4/TAX-2 heteromeric channels

To examine the functional properties of TAX-2, we transfected HEK293 cells with a TAX-2 expression plasmid, followed by excised inside–out patch recording. Co-transfection with a green fluorescent protein (GFP) expression plasmid was performed to identify cells likely to express the channel [17]. We were unable to detect any cyclic nucleotide-activated conductances from the GFP-expressing cells.

It has been proposed that TAX-4 and TAX-2 function as a heteromeric channel in vivo 7, 17. To test

Discussion

tax-4 and tax-2 mutants show similar defects in sensory behaviors. The mutants fail to respond to temperature, salts, and AWC-sensed odorants such as benzaldehyde, 2-butanone, and isoamyl alcohol. GFP-tagged functional TAX-4 and TAX-2 proteins are expressed in an almost completely overlapping subset of sensory neurons, including those that mediate the relevant sensory behaviors, and both of them are localized at sensory cilia. Furthermore, overexpression of the tax-4 gene rescues the defects of

Acknowledgements

This work was supported by grants from Japan Society for the Promotion of Science (JSPS) (to H.K.), the HFSPO (to I.M. and C.I.B.) and PRESTO (to I.M.), Science and Technology Agency of Japan and Japan Society for the Promotion of Science (Research for the Future 97L00401) (to Y.O.), and the Ministry of Education, Science, Sports and Culture of Japan to I.M. and Y.O. H.K. is a recipient of the scholarship from JSPS. N.L'E. is a fellow of the Howard Hughes Medical Institute and C.I.B. is an

References (30)

  • M.D Varnum et al.

    Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels

    Neuron

    (1995)
  • C.D Benham et al.

    A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle

    Nature

    (1987)
  • J Bradley et al.

    Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP

    Proc. Natl. Acad. Sci. USA

    (1994)
  • J Bradley et al.

    Functional expression of the heteromeric `olfactory' cyclic nucleotide-gated channel in the hippocampus: a potential effector of synaptic plasticity in brain neurons

    J. Neurosci.

    (1997)
  • M.J Caterina et al.

    The capsaicin receptor: a heat-activated ion channel in the pain pathway

    Nature

    (1997)
  • Cited by (0)

    1

    Present address: Division of Biological Sciences, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.

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