Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed Icon. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the Icon channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.
Key words NIH 3T3 9 Regulatory volume decrease 9 Chloride channels -Ian " Nucleotide block 9 Antisense events essential for cell volume regulation after swelling [16,21]. The identification of the proteins involved and their structure-function relations is imperative for understanding this critical cell regulatory mechanism. Cell swelling leads to the activation of anionic currents after overexpression of P-glycoprotein or C1 C-2 [7, 10, 27], two cloned proteins believed to be involved in chloride transport. These two proteins, however, do not seem to participate in our system, since the current measured after swelling NIH 3T3 fibroblasts (Ic1, see below) is insensitive to verapamil, a drug known to block P-glycoprotein; moreover, the kinetics of Ic~ (and the outwardly rectifying chloride current of Icln, see below) are dissimilar to those of C1C-2, as is the selectivity for different anions I->Br-~> C1-for Icon [23] versus C1-~>Br->I-for C1C-2 [10]). It has been shown that a recently cloned protein leads to marked outwardly rectifying chloride current (Icln) after overexpression in Xenopus laevis oocytes [22]. This protein was cloned from Madin Darby canine kidney (MDCK) cells which are able to effectively regulate their volume after swelling [21,31]. Rectification, kinetics and selectivity of Icon are reminiscent of chloride currents elicited in T84 and primary culture epithelial cells after reduction of extracellular osmolarity [25,32].