The Tyrosine Kinase p56lck Mediates Activation of Swelling-induced Chloride Channels in Lymphocytes

Abstract

Osmotic cell swelling activates Cl− channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl−swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl−swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl−swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, 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 p56lck. We conclude that osmotic swelling activates ICl−swell in lymphocytes via the tyrosine kinase p56lck.

Calcium Mobilization Evoked by Hepatocellular Swelling Is Linked to Activation of Phospholipase Cγ

“…If FAK is involved in volume-sensitive PLC␥ activation, our observations would suggest that focal adhesions may not be the sole site of the swelling-activated interaction between these two proteins because the bulk of plasma membrane-bound tyrosinephosphorylated PLC␥, as detected by immunofluorescence, did not appear to be present in focal adhesions during times corresponding to maximal swelling-induced tyrosine phosphorylation of PLC␥ as determined by immunoblot analysis. Members of the Src family of tyrosine kinases, which can associate with PI 3-kinase (29) as well as FAK (30), mediate volume recovery from swelling in lymphocytes (31) and play important roles in mechanotransduction (32). In lung, mechanical strain elicits Src-mediated PLC␥ activation as a consequence of rearrangements of the actin cytoskeleton (33).…”

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…20,28 In addition, signaling pathways contributing to VRAC activity include phosphatidyl-inositol-3-phosphate kinase (PI3K), NADH-dependent oxidases and reactive oxygen species (ROS) as well as tyrosine kinases. [29][30][31][32][33][34][35][36] Recently it was demonstrated that knockdown of integrin b 1 reduces RVD in adherent Ehrlich Lettre ascites cells (ELA) cells 37 and as integrin b 1 stretch in Figure 1. VRAC characteristics.…”

Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis

“…To drive the net efflux of water, which leads to cell shrinkage, release of anions should take place in parallel with K ϩ release because of the restraint of electroneutrality. Stimulation of CD95 (Fas) receptor has actually been shown to induce activation of outwardly rectifying Cl Ϫ channels (8), which were also activated by osmotic swelling (9), in lymphoid Jurkat cells. Thus, there exists a possibility that volume regulation mechanisms (10)(11)(12), including volume-regulatory Cl Ϫ and K ϩ channels, are disordered, thereby inducing the AVD during the apoptotic process.…”