What are Tetrahymena? Eukaryotic, single cell, ciliated, motile About 50 to 70um long Excitable (action potentials) Model sensory cell (chemosensory, thermosensory,

What are Tetrahymena?

• Eukaryotic, single cell, ciliated, motile• About 50 to 70um long• Excitable (action potentials)• Model sensory cell (chemosensory,

thermosensory, mechanosensory)• Grow to 500,000 cells/ml as clonal,

axenic cultures• Electrophysiology, biochemistry,

behavior and molecular biology well described

• Genome sequenced, knockouts

• How do they respond to external GTP and ATP?

Tetrahymena responses to a GTP gradient (2ul of 10mM GTP added on the right)

Why Avoid External ATP and GTP?

ATP and GTP are Depolarizing Chemorepellents.

• They are supposed to be INSIDE cells• They are released during cell death and lysis• Therefore, they represent lysis of nearby cells• They are cytoplasmic indicators• “Blood-in-the-water” signals for dangerous

situations (worth avoiding)• Negative necrotaxis (necrophobiac)• Choices: Avoid, adapt or die• How do you study these responses in Tetrahymena?

Approaches for Studying ATP and GTP Responses in Tetrahymena

1. Behavioral bioassays

2. Intracellular electrophysiology

3. External 32P-ATP and 32P-GTP binding

4. External photoaffinity labeling

5. Pharmacology

6. Genome database mining

7. Forward Genetics: Behavioral mutant screens

8. Reverse Genetics: Gene knockouts

9. Proteomics: Identification of gene product functions in the transduction pathways

What is the Behavioral Bioassay?

Single cell added to solution containing ATP or GTP

Different cell added to control solution

Cells in micropipet

Cells in micropipet

Cells show “avoiding reactions” (AR)

No AR, straight swimming

Tetrahymena Behavioral Responses to ATP and GTP

What are the Electrophysiological Responses

to ATP and GTP?

Electrophysiological Responses of Tetrahymena to Repellents

Transient receptor potentials last about 1 minute. Graded action potentials seen as fast spikes

Do Tetrahymena adapt to ATP and GTP?

Chemosensory adaptation is a decrease in responsiveness to a ligand as a function of

time of exposure to that stimulus

No Cross-Adaptation between ATP and GTP Responses

How Can We Assay External ATP and GTP Binding?

Does this binding change during adaptation?

Adaptation Decreases In Vivo Radioactive 32P-GTP Binding

Non-adaptedDe-adapted

Adapted

Same thing happens with ATP too

32P Azido ATP and GTP Photoaffinity LabelingATP GTP

An externally facing ATP-binding protein was separated by standard SDS-PAGE and visualized by autoradiography. A GTP-binding protein band was separated by 2D SDS-PAGE and also visualized by autoradiography

Evidence for Different ATP and GTP Receptors

• Cold GTP doesn’t compete with hot ATP for binding (and vice-versa)

• No cross-adaptation (behavior and binding)• ATP responses are inhibited by pertussis toxin,

calphostin C and Rp-cAMPS but not GTP responses• The ATP receptor may be metabotropic (P2Y-like?) and

the GTP receptor ionotropic (novel?)• 32P-photoaffintity labeling shows 58kD ATP binding

protein and 48kD GTP binding protein

Tetrahymena Genome Database Comparisons

• Highest homolgies found to P2Y type mammalian (58% similarity) and 7-transmembrane receptor from Arabidopsis (42% similarity)

• High homology of ecto-ATPase to mammalian ecto-ATPases (44% similarity)

• Many signal transduction genes present (G-proteins, protein kinase C, tyrosine kinase, calmodulin, cAMP-dependent protein kinase, etc.)

What Stuctural Information Can be Predicted from Database Sequences?

Hydropathy Plot and Transmembrane Regions of rat P2Y2 Receptor (P49651)

Looks like a 7-transmembrane receptor

Hyropathy Plot and Transmemebrane Regions of Tetrahymena TP2Y

Looks like a 7-transmembrane receptor

Predicted Transmembrane Domains of Rat P2Y2 and TP2Y

Rat P2Y2

Tetrahymena TP2Y

Although different amino acid sequences, they look structurally quite similar

Structure Determines Function

Example: Structure of

ATP

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