Abstract
Although G-protein coupled receptors (GPCRs) are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490). Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba2+ and K+, suggesting a decrease in basal excitability (decrease in Ca2+ channel activity). The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA) and proteose peptone (PP), two known chemoattractants in Tetrahymena. Using microsomal [35S]GTPγS binding assays, we found that wild-type (CU427) have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor), addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes) towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein.
Highlights
The ciliated protozoan Tetrahymena thermophila shows chemosensory responses to many different stimuli but no chemoreceptors have been fully verified from gene to ligand
Nine candidate G-protein coupled receptors (GPCRs) were selected from the Tetrahymena Genome Database (TGD, http://www.ciliate.org/)
We propose that Gpcr6p is required for a constitutive signal that is integral in maintaining proper basal swimming behavior in Tetrahymena
Summary
The ciliated protozoan Tetrahymena thermophila shows chemosensory responses to many different stimuli but no chemoreceptors have been fully verified from gene to ligand. As free-swimming cells, Tetrahymena change their swim speed and swimming direction in response to many types of chemorepellents [1,2,3,4] and chemoattractants [5,6,7,8]. The general model from studies of the related ciliate, Paramecium, is that chemoattractants cause somatic hyperpolarization, faster forward swimming speed, and less directional changes [9]. As intraciliary free Ca2+ rises, the beat frequency slows and when the free Ca2+ exceeds 1026 M, the cilia reverse their direction of beat [10,11] These unicells integrate sensory information in the form of changes in membrane potentials to generate an appropriate ciliary response. The intracellular electrophysiological measurements in Tetrahymena have shown that they are generally similar to those of Paramecium, establishing Tetrahymena as a suitable tool for studies of membrane excitation and chemosensory transduction mechanisms [12,13]
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