Transient receptor potential-like channels are essential for calcium signaling and fluid transport in a Drosophila epithelium.
Calcium signaling is an important mediator of neuropeptide-stimulated fluid transport by Drosophila Malpighian (renal) tubules. We demonstrate the first epithelial role, in vivo, for members of the TRP family of calcium channels. RT-PCR revealed expression of trp, trpl, and trpgamma in tubules. Use of antipeptide polyclonal antibodies for TRP, TRPL, and TRPgamma showed expression of all three channels in type 1 (principal) cells in the tubule main segment. Neuropeptide (CAP(2b))-stimulated fluid transport rates were significantly reduced in tubules from the trpl(302) mutant and the trpl;trp double mutant, trpl(302);trp(343). However, a trp null, trp(343), had no impact on stimulated fluid transport. Measurement of cytosolic calcium concentrations ([Ca(2+)](i)) in tubule principal cells using an aequorin transgene in trp and trpl mutants showed a reduction in calcium responses in trpl(302). Western blotting of tubule preparations from trp and trpl mutants revealed a correlation between TRPL levels and CAP(2b)-stimulated fluid transport and calcium signaling. Rescue of trpl(302) with a trpl transgene under heat-shock control resulted in a stimulated fluid transport phenotype that was indistinguishable from wild-type tubules. Furthermore, restoration of normal stimulated rates of fluid transport by rescue of trpl(302) was not compromised by introduction of the trp null, trp(343). Thus, in an epithelial context, TRPL is sufficient for wild-type responses. Finally, a scaffolding component of the TRPL/TRP-signaling complex, INAD, is not expressed in tubules, suggesting that inaD is not essential for TRPL/TRP function in Drosophila tubules.
MacPherson
Matthew R
MR
IBLS Division of Molecular Genetics, University of Glasgow, UK. s.a.davies@bio.gla.ac.uk
Pollock
Valerie P
VP
Kean
Laura
L
Southall
Tony D
TD
Giannakou
Maria E
ME
Broderick
Kate E
KE
Dow
Julian A T
JA
Hardie
Roger C
RC
Davies
Shireen A
SA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001692:Biological Transport; D001826:Body Fluids / Q000502:physiology*; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000502:physiology; D003434:Crossing Over, Genetic; D017931:DNA Primers; D004330:Drosophila / Q000502:physiology*; D029721:Drosophila Proteins / Q000235:genetics / Q000502:physiology; D005260:Female; D008297:Male; D008317:Malpighian Tubules / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050051:Transient Receptor Potential Channels
D000906:Antibodies; D015220:Calcium Channels; D002148:Calmodulin-Binding Proteins; D017931:DNA Primers; D029721:Drosophila Proteins; D008565:Membrane Proteins; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila