Characterization of a chloride current in the larval epidermis of the beetle Tenebrio molitor

Voltage-clamp analysis of single cuticle-attached epidermal cells dissected from the newly-ecdysed mealworm revealed the presence of a large inwardly- rectifying anion (i.e. outwardly-going) current. In many cells this current formed spontaneously on breaking into the cell with the patch pipette when the bath solution was isoosmotic with the pipette solution (415 mosmol/l). The current was evoked rapidly by electrical stimulation or by bathing the cells in hyposmotic saline (335 mosmol/l). The reversal potential of the a ctivated current shifted in agreement with the Nernst prediction for Cl- wh en the transmembrane chloride gradient was altered by partially substitutin g bath or patch pipette Cl- with gluconate(-). Substitution of Na+ with cho line(+) or K+ with TEA and Ba+ in the bath or pipette solutions did not alt er the reversal potential. Addition of 200 mu mol/l cyclic AMP or 1 mmol/l cyclic GMP to the pipette solution increased the initial current strength a nd reduced the time taken to reach half peak amplitude from 117 sec to 49 s ec and 41 sec, respectively. Cyclic AMP also raised the threshold at which the current developed under hyperosmotic conditions by about 20 mosmol/l. A ddition of the Cl- channel blockers diphenylamine-2-carboxylic acid (200 mu mmol/l) and diisothiocyanostilbene-2,2'-disulphonic acid (250 mu mol/l) to the bath solution reduced the inwardly-rectifying anion current by 50%. Th is current was barely detectable in cells prepared from the mid-instar inte gument. This non-constitutive pattern of expression suggests that cellular Cl- efflux (and that of other anions) may be required during moult-cycle sp ecific processes such as moulting fluid formation and cell volume regulatio n. As the strength of the epidermal anion current could be raised by the ex ogenous application of cytosolic cyclic nucleotides, the activity of the an ion channels responsible for this current may normally be regulated by yet- to-be-identified hormone(s) or neuropeptide(s) acting on this tissue. (C) 1 999 Elsevier Science Ltd. All nights reserved.