Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. Here, we resolve this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a-mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a-independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia.

A major class of nicotinic receptors in the nervous system is one that binds ␣-bungarotoxin and contains the ␣7 gene product. PC12 cells, frequently used to study nicotinic receptors, express the ␣7 gene and have binding sites for the toxin, but previous attempts to elicit currents from the putative receptors have failed. Using whole-cell patch-clamp recording techniques and rapid application of agonist, we find a rapidly desensitizing acetylcholine-induced current in the cells that can be blocked by ␣-bungarotoxin. The current amplitude varies dramatically among three populations of PC12 cells but correlates well with the number of toxin-binding receptors. In contrast, the current shows no correlation with ␣7 transcript; cells with high levels of ␣7 mRNA can be negative for toxin binding and yet have other functional nicotinic receptors. Northern blot analysis and reverse transcription-PCR reveal no defects in ␣7 RNA from the negative cells, and immunoblot analysis demonstrates that they contain full-length ␣7 protein, although at reduced levels. Affinity purification of toxin-binding receptors from cells expressing them confirms that the receptors contain ␣7 protein. Transfection experiments demonstrate that PC12 cells lacking native toxin-binding receptors are deficient at producing receptors from ␣7 gene constructs, although the same cells can produce receptors from other transfected gene constructs. The results indicate that nicotinic receptors that bind ␣-bungarotoxin and contain ␣7 subunits require additional gene products to facilitate assembly and stabilization of the receptors. PC12 cells offer a model system for identifying those gene products.

The Malpighian (renal) tubule of Drosophila melanogaster is a useful model for studying epithelial transport. The purpose of this study was to identify factors responsible for modulating transepithelial chloride conductance in isolated tubules. I have found that tyrosine and several of its metabolites cause an increase in chloride conductance. The most potent of these agonists is tyramine, which is active at low nanomolar concentrations; the pharmacology of this response matches that of the previously published cloned insect tyramine receptor. In addition, the tubule appears capable of synthesizing tyramine from applied tyrosine, as shown by direct measurement of tyrosine decarboxylase activity. Immunohistochemical staining of tubules with an antibody against tyramine indicates that the principal cells are the sites of tyramine production, whereas previous characterization of the regulation of chloride conductance suggests that tyramine acts on the stellate cells. This is the first demonstration of a physiological role for an insect tyramine receptor.

for preparing the ciliary ganglion cell cultures, and Brtan Scott for preparing the Xenopcrs oocytes. The chicken a7 full-length construct used to generate RNA for expression i n oocytes was kindly provi ded by Dr. Ralf Schoepfer (Untversity of Heidelberg).

When expressed in the Xenopus oocyte, the minK protein induces a slowly activating voltage-dependent potassium current (Isk). We studied the modulation of this current by altering intracellular cAMP levels and found that the amplitude of Isk is dramatically increased by treatments that raise cAMP levels and decreased by agents that lower cAMP levels. Preinjection of a protein inhibitor of the cAMP-dependent protein kinase blocked the effects of increased cAMP levels. There were no changes in the voltage dependence or kinetics of Isk. Mutations that eliminate a potential phosphorylation site on the minK protein did not block the effects of activating the kinase. In addition, the membrane capacitance of the oocyte increased and decreased in parallel with Isk. Our results fit a mechanism in which channel proteins are selectively inserted into and removed from the plasma membrane in response to changes in kinase activity.

Mutations in the Drosophila gene drop-dead (drd) result in early adult lethality and neurodegeneration, but the molecular identity of the drd gene and its mechanism of action are not known. This paper describes the characterization of a new X-linked recessive adult-lethal mutation, originally called lot’s wife (lwf1) but subsequently identified as an allele of drd (drdlwf); drdlwf mutants die within two weeks of eclosion. Through mapping and complementation, the drd gene has been identified as CG33968, which encodes a putative integral membrane protein of unknown function. The drdlwf allele is associated with a nonsense mutation that eliminates nearly 80% of the CG33968 gene product; mutations in the same gene were also found in two previously described drd alleles. Characterization of drdlwf flies revealed additional phenotypes of drd, most notably, defects in food processing by the digestive system and in oogenesis. Mutant flies store significantly more food in their crops and defecate less than wild-type flies, suggesting that normal transfer of ingested food from the crop into the midgut is dependent upon the DRD gene product. The defect in oogenesis results in the sterility of homozygous mutant females and is associated with a reduction in the number of vitellogenic egg chambers. The disruption in vitellogenesis is far more severe than that seen in starved flies and so is unlikely to be a secondary consequence of the digestive phenotype. This study demonstrates that mutation of the drd gene CG33968 results in a complex phenotype affecting multiple physiological systems within the fly.

The minK gene encodes a protein of 130 amino acids that has a single transmembrane segment and is expressed in many tissues including heart, uterus, and kidney. When Xenopus oocytes are injected with minK mRNA, a very slowly activating voltage-dependent potassium current is induced in these cells. The induced channels appear to result from the interaction of the minK protein with other channel-forming subunits such as the KvLQT1 channel. The minK protein is intimately associated with the structure of the resultant channels, and mutations in minK can alter ion selectivity and modulation by second messengers. Strong candidates for native currents regulated by the minK protein include the slow component of the cardiac delayed rectifier and potassium currents recorded across epithelial cells in vestibular organs and cochlea.

The minK protein induces a slowly activating voltage-dependent potassium current when expressed in Xenopus oocytes. In order to measure the levels of minK protein in the plasma membrane, we have modified the minK gene by inserting a 9 amino acid epitope into the N- terminal domain of the protein sequence. When intact live oocytes are injected with the modified minK RNA and subsequently incubated with an antibody to this epitope, specific binding is detected, indicating that the N-terminal domain is extracellular. We found that when oocytes are injected with amounts of minK mRNA up to 50 ng, the levels of protein at the surface are proportional to the amount of injected mRNA. In contrast, the amplitude of the minK current recorded in the oocytes saturates at 1 ng of injected mRNA. Although the amplitude of the currents is not altered by increasing mRNA levels above 1 ng, the kinetics of activation of the current differ in oocytes with high or low levels of minK RNA. In particular, activation is slower with higher levels of minK protein in the plasma membrane. Finally, we find that increasing intracellular cAMP levels, which increases the amplitude of minK currents, does not alter surface expression of the minK protein but produces a small increase in the rate of activation of the current. Our results support a model in which minK protein forms functional potassium channels by association with a factor endogenous to the oocyte.