Properties of single Drosophila Trpl channels expressed in Sf9 insect cells.
The transient receptor potential (trp)-like (trpl) gene is thought to encode an ion channel important for signal transduction in Drosophila photoreceptor cells. Consistent with this hypothesis, heterologous expression of the trpl-encoded protein (Trpl) is associated with the appearance of an outwardly rectifying, nonselective cation current. In the present study, single channels were recorded in cell-attached, inside-out, and outside-out membrane patches from Sf9 insect cells infected with recombinant baculovirus-containing trpl cDNA under control of the polyhedrin promoter. The single-channel current-voltage relationship was linear from -100 to +80 mV with a slope conductance of 89-110 pS. The probability of opening was voltage sensitive, increasing at positive potentials contributing to the outwardly rectifying properties of the whole cell currents. The single channels 1) were never observed in Sf9 cells infected with recombinant baculovirus containing the B2 bradykinin receptor cDNA or in noninfected Sf9 cells; 2) appear at the same time postinfection as the Trpl whole cell current; 3) were nonselective with respect to Na+, Ca2+, and Ba2+; 4) were blocked by 1-2 mM La3+ and Gd3+ (but not 10 microM); and 5) were blocked by 4-8 mM Mg2+. The single Trpl channel activity increased spontaneously with time after patch formation, and the activity was further increased by application of bradykinin to cells expressing both the B2 bradykinin receptor and the Trpl protein. These results suggest that this single-channel activity reflects expression of the Trpl protein and provides conclusive evidence that trpl encodes a nonselective cation channel consistent with its proposed role in Drosophila phototransduction.
Kunze
D L
DL
Rammelkamp Center for Education and Research, Case Western Reserve University, Cleveland, Ohio 44109, USA.
Sinkins
W G
WG
Vaca
L
L
Schilling
W P
WP
D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D002460:Cell Line; D004330:Drosophila; D029721:Drosophila Proteins; D004594:Electrophysiology; D005682:Gadolinium / Q000494:pharmacology; D007313:Insecta; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000502:physiology*; D007811:Lanthanum / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000502:physiology*; D018408:Patch-Clamp Techniques; D018002:Receptors, Bradykinin / Q000502:physiology; D013997:Time Factors; D050051:Transient Receptor Potential Channels
D002148:Calmodulin-Binding Proteins; D029721:Drosophila Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D018002:Receptors, Bradykinin; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila; D007811:Lanthanum; D005682:Gadolinium; D008274:Magnesium