lck Mediates Activation of Swelling-induced Chloride Channels in Lymphocytes

Osmotic cell swelling activates C l 2 channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56 lck , a src -like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (I C l 2 swell ) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of I C l 2 swell is defective in p56 lck deficient cells. Retransfection of p56 lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of I C l 2 swell . Addition of purified p56 lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56 lck washed into the cytoplasm activates I C l 2 swell in native and p56 lck -deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56 lck , slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56 lck . We conclude that osmotic swelling activates I C l 2 swell in lymphocytes via the tyrosine kinase p56 lck . W hen cells are exposed to hypotonic stress, initial swelling is followed by regulatory volume decrease (RVD). 1 In lymphocytes, RVD involves activation of volume-sensitive anion channels, leading to membrane depolarization and thereby opening voltageactivated potassium channels (Deutsch and Lee, 1988). This produces a net loss of KCl, resulting in regulatory decrease of intracellular osmolarity and driving H 2 O out of the cell. Anion channels activated by osmotic stress are expressed in a wide variety of nonexcitable and excitable tissues (for review see Lang et al., 1997), and are thought to mediate an efflux of osmotically active anions in response to increased cellular volume (Cahalan and Lewis, 1988). Biophysical and pharmacological differences have been observed between swelling-activated anion channels found in lymphocytes and other tissues (Kunzelmann et al., 1989; Solc and Wine, 1991; Sorota, 1992; Lewis et al., 1993). Most of these channels are outwardly rectifying and possess unitary conductances ranging from 20 to 90 pS. At least three different anion channels have been characterized at the single channel level in membrane patches from lymphocytes (Lewis and Cahalan, 1988; Nishimoto et al., 1991; Garber, 1992). However, a clear relationship between single-channel currents and whole-cell currents is lacking. Although the genes for a number of different Cl 2 channel proteins have been cloned, the proteins forming swelling activated anion channels in lymphocytes have not yet been identified. Their activation mechanism is even less completely understood. In lymphocytes, neither an increase in cytosolic calcium nor in membrane area is necessary for activation of volume-sensitive anion channels (Ross et al., 1994; Ross and Cahalan, 1995). However, participation of cytoskeletal elements in channel activation has been demonstrated (Levitan et al., 1995). Most interestingly, the presence of intracellular ATP is necessary for maintenance or repeated activation of the swelling-activated anion current (Lewis et al., 1993; Ross et al., 1994). Recent work has shown inhibition of volume-sensitive chloride current in heart cells by tyrosine-kinase inhibitors (Sorota, 1995) and regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel by tyrosine phosphorylation (Fischer and Machen, 1996). In this study we have examined the role of a src -like lymphocyte tyrosine kinase, p56 lck , in the activation of swellingactivated anion channels in lymphocytes. E. Gulbins and F. Lang contributed equally to this work. Address all correspondence to Albrecht Lepple-Wienhues, Physiology I, University of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany. Tel.: (49) 7071-2975285. Fax: (49) 7071-293073. E-mail: [email protected] 1. Abbreviations used in this paper : RVD, regulatory volume decrease; I Cl , chloride current; I Cl 2 swell , swelling-activated chloride current; CFTR, cystic fibrosis transmembrane conductance regulator; DIDS, diisothiocyanato-2-2-stilbenesulfonic acid. on A uust 4, 2017 jcb.rress.org D ow nladed fom