Tyrosine Phosphorylation-dependent Suppression of a Voltage-gated K+ Channel in T Lymphocytes upon Fas Stimulation

Selective cell death plays a critical role in the development of the immune system and in the elimination of target cells expressing foreign antigens. Most of programmed cell death occurs by apoptosis. Apoptotic cell death of lymphocytes can be triggered by ligation of APO-1/Fas (CD95) antigen (Suda, T., and Nagata, S. (1994) J. Exp. Med. 179, 873-879; Nagata, S., and Golstein, P. (1995) Science 267, 1449-1456). We find that activation of Fas leads to the inhibition of the voltage-dependent n-type K+ channels (Kv1.3) studied by patch clamp technique in Jurkat T lymphocytes. Tyrosine kinases have been shown to be crucial in Fas-induced cell death (Eischen, C. M., Dick, C. J., and Leibson, P. J. (1994) J. Immunol. 153, 1947-1954). The inhibition of the current is correlated with the tyrosine phosphorylation of immunoprecipitated and blotted K+ channel protein. We show, that the Src-like protein-tyrosine kinase inhibitor herbimycin A and the deficiency of the p56lck tyrosine kinase in mutant Jurkat cells abolished the channel inhibition and phosphorylation by anti-Fas antibody, while reconstitution of the p56lck kinase partly restored these effects of Fas receptor triggering. These results suggest a regulation of n-type K+ channels by tyrosine kinases upon Fas receptor triggering, which might be important for apoptosis. Selective cell death plays a critical role in the development of the immune system and in the elimination of target cells expressing foreign antigens. Most of programmed cell death occurs by apoptosis. Apoptotic cell death of lymphocytes can be triggered by ligation of APO-1/Fas (CD95) antigen (Suda, T., and Nagata, S. (1994) J. Exp. Med. 179, 873-879; Nagata, S., and Golstein, P. (1995) Science 267, 1449-1456). We find that activation of Fas leads to the inhibition of the voltage-dependent n-type K+ channels (Kv1.3) studied by patch clamp technique in Jurkat T lymphocytes. Tyrosine kinases have been shown to be crucial in Fas-induced cell death (Eischen, C. M., Dick, C. J., and Leibson, P. J. (1994) J. Immunol. 153, 1947-1954). The inhibition of the current is correlated with the tyrosine phosphorylation of immunoprecipitated and blotted K+ channel protein. We show, that the Src-like protein-tyrosine kinase inhibitor herbimycin A and the deficiency of the p56lck tyrosine kinase in mutant Jurkat cells abolished the channel inhibition and phosphorylation by anti-Fas antibody, while reconstitution of the p56lck kinase partly restored these effects of Fas receptor triggering. These results suggest a regulation of n-type K+ channels by tyrosine kinases upon Fas receptor triggering, which might be important for apoptosis. INTRODUCTIONThe programmed cell death is a process characterized by DNA fragmentation and apoptotic cell morphology. Apoptosis can be triggered by a variety of extrinsic and intrinsic signals, including ligation of APO-1/Fas (CD95) antigen or purified Fas ligand (1Suda T. Nagata S. J. Exp. Med. 1994; 179: 873-879Crossref PubMed Scopus (499) Google Scholar, 2Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3965) Google Scholar). One of the crucial events in the early phase of Fas-induced apoptosis is the tyrosine phosphorylation of multiple cellular proteins (3Eischen C.M. Dick C.J. Leibson P.J. J. Immunol. 1994; 153: 1947-1954PubMed Google Scholar). Incubation of lymphocytes with herbimycin A, an inhibitor of Src-like tyrosine kinases prevents Fas-induced apoptosis (3Eischen C.M. Dick C.J. Leibson P.J. J. Immunol. 1994; 153: 1947-1954PubMed Google Scholar).Thus far, nothing is known about the possible involvement of ion channels in apoptotic cell death. The present study has been performed to test for an influence of Fas stimulation on ion channel activity. As a result, Fas indeed led to a striking inhibition of n-type K+ channels, the most abundant K+ channels in lymphocytes. To further elucidate the mechanism of this inhibition, we looked for phosphorylation of the channel protein following Fas stimulation. Triggering of the Fas receptor was indeed followed by tyrosine phosphorylation of the channel. In order to evaluate the significance of the phosphorylation for channel inhibition, we have performed studies examining the effect of Fas on n-type K+ channel activity in cells deficient of tyrosine kinase and in the presence of tyrosine kinase inhibitor.In the present study we show that the n-type K+ channels are inhibited upon Fas stimulation of Jurkat T lymphocytes and that the suppression of current is correlated with tyrosine phosphorylation of the channel protein by the p56lck tyrosine kinase.RESULTSPatch clamp experiments were performed to assess the effect of Fas stimulation on macroscopic potassium current and single potassium channel activity in Jurkat human T lymphocytes. As shown in Fig. 1A, whole-cell outward currents, elicited at depolarizing voltages, exhibited kinetics typical for n-type K+ channels (10Matteson D.R. Deutsch C. Nature. 1984; 307: 468-471Crossref PubMed Scopus (233) Google Scholar, 11De Coursey T.E. Chandy K.G. Gupta S. Cahalan M.D. Nature. 1984; 307: 465-468Crossref PubMed Scopus (587) Google Scholar, 12Cahalan M.D. Chandy K.G. De Coursey T.E. Gupta S. J. Physiol. (Lond.). 1985; 358: 197-237Crossref Scopus (322) Google Scholar, 13Deutsch C. Krause D. Lee S.C. J. Physiol. (Lond.). 1986; 372: 405-423Crossref Scopus (120) Google Scholar). According to ion substitution experiments, the current was K+-selective (a shift of reversal potential to 8 ± 6 mV was observed with 140 mM K+ bath medium, n = 4). Furthermore, the current was sensitive to charybdotoxin, a well known inhibitor of voltage-gated K+ channels (14Sands S.B. Lewis R.S. Cahalan M.D. J. Gen. Physiol. 1989; 93: 1061-1074Crossref PubMed Scopus (125) Google Scholar). Jurkat cells, displaying a stable current in whole-cell configuration for at least 5 min, were used either as time controls (n = 18) or for stimulation (n = 15). Cells were stimulated with a total amount of 1 µg of antibody by perfusing the chamber with 200 ng/ml monoclonal anti-human Fas antibody (anti-Fas) 1The abbreviation used is: anti-Fasmonoclonal anti-human Fas antibody. (n = 10) or with 200 ng/ml polyclonal anti-mouse Fas antibody (n = 5) (Fig. 1B). For time controls, the cells were left untreated (n = 18) (Fig. 1B). On Fig. 1B, zero time corresponds to the beginning of incubation. Stimulation via anti-human Fas treatment resulted in a 46 ± 4% decrease of the whole-cell peak current within 15 min, while incubation of the human cells with anti-mouse Fas antibody caused a 4 ± 2% decrease over the same period (Fig. 1, B and C). Stimulation with anti-CD20 antibody was similarly ineffective (n = 3) (not shown). These latter experiments have been performed in order to test the specificity of anti-human Fas antibody's effect on channel activity. The time course and the extent of the effect of the antibody was independent of whether it was added after 9-12 or 23-25 min of recording in whole-cell configuration. The current significantly decreased for all voltages ≥10 mV (Fig. 1, A and D). The current suppression was not due to changes in the voltage-dependent gating properties of the channel, since the intrinsic voltage sensitivity of the channel was not altered. The inhibition was not reversible within 20 min respective to the end of the stimulation (n = 2). The remaining current after inhibition by the antibody was completely abolished by 50 nM charybdotoxin (n = 3), indicating that the pharmacological properties of the channel were not altered.Inhibition of K+ channels by Fas triggering was also observed at single-channel levels in outside-out patches (Fig. 2A) (n = 3). These experiments indicated that anti-Fas antibody inhibited the whole-cell current by decreasing the open probability of the channels without affecting significantly the single-channel amplitude. Fig. 2B shows representative current traces from an unstimulated patch indicating that the channels' activity was stable (n = 4), while Fig. 2C demonstrates that incubation of a patch with anti-mouse Fas antibody did not alter the channels' open probability (n = 3).Fig. 2Effect of Fas stimulation on activity at the single-channel level. A, outward currents, elicited by 5-s depolarizing steps to 0 mV from −70 mV holding potential at 2-min intervals in an outside-out patch. Upper trace, activity in control condition; lower two traces, activity from the same patch after 6 and 9 min of incubation with 200 ng/ml antibody, respectively. Dashed lines indicate the zero-current level. The open probability (Po) of the channels decreased by 30% and 67%, respectively. The Po was calculated by integrating the current through open channels in a 5-s segment and dividing the integral by the time of recording multiplied by the current through a single channel (1.5 pA) and the total number of channels observed in the patch. B, n-K+ channel activity in a 5-s segment, elicited by the pulse protocol described in A, in control conditions. Upper trace, activity of the first pulse step in an outside-out patch; lower trace, activity from the same patch 12 min later. In this experiment, the open probability of the channel varied between 0.76 and 0.92 during the subsequent depolarizing steps, and the activity showed no rundown. Original traces are representative of 4 similar experiments. C, outward currents, elicited by the pulse protocol described in A, in control condition (upper trace) and after 18 min of incubation with anti-mouse Fas antibody (lower trace), from the same patch. No decrease in the channels' open probability could be observed during the experiment (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To gain insight into the mechanism of the inhibition of the n-type K+ channel, we examined whether the suppression of the current is correlated with tyrosine phosphorylation of the K+ channel protein. Immunoprecipitation and blotting of the Kv1.3 channel protein from Jurkat cells revealed tyrosine phosphorylation of the 65-kDa n-type K+ channel protein following stimulation of the cells via Fas (Fig. 3A). Tyrosine phosphorylation was observed for at least 20 min.Fig. 3Kv1.3 channel protein is tyrosine-phosphorylated upon Fas stimulation. A: upper panel, tyrosine phosphorylation of the 65-kDa Kv1.3 protein after incubation of Jurkat cells with anti-Fas antibody for the indicated times; lower panel, stripping and reprobing the blot with anti-Kv1.3 antiserum reveals similar protein amounts in all lanes. Unspecific immunoprecipitates (up) were performed with prebleed. B: upper panel, tyrosine phosphorylation of immunoprecipitated and blotted channel protein in Jurkat, herbimycin A (10 µM)-treated Jurkat (HA), JCaM 1.6 (JCaM), and p56lck-reconstituted JCaM 1.6 (JCaM+lck) cells after incubation with anti-Fas antibody for the indicated times; lower panel, similar concentrations of proteins were loaded in all lanes, as shown by stripping and reprobing the blot with anti-Kv1.3 antiserum.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Herbimycin A (10 µM), an inhibitor of the Src-like tyrosine kinases known to inhibit Fas-induced apoptosis, prevented tyrosine phosphorylation of Kv1.3 protein (Fig. 3B). Likewise, herbimycin A attenuated the inhibition of the whole-cell n-type K+ current by anti-human Fas in Jurkat cells (Fig. 3B).Since p56lck is the main non-receptor, membrane-bound Src-like tyrosine kinase in Jurkat T cells, we tested for Fas-induced n-type K+ channel suppression in p56lck-deficient Jurkat cells (JCaM 1.6) (4Straus D.B. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (924) Google Scholar). As illustrated on Fig. 3B, tyrosine phosphorylation of the channel protein was completely absent in JCaM 1.6 cells. Moreover, the reconstitution of these cells with p56lck restored tyrosine phosphorylation of the channel protein upon Fas stimulation. Western blot analyses (Fig. 3, A and B, lower panels) indicated that the differences in the extent of tyrosine phosphorylation in the various cell lines was not simply due to different Kv1.3 protein concentrations in the immunoprecipitates.The lack of tyrosine phosphorylation of n-type K+ channels in JCaM 1.6 cells following exposure to anti-human Fas is paralleled by the near complete absence of n-type K+ channel inhibition after Fas receptor stimulation (Fig. 4, A and B). However, the n-type K+ channels in JCaM 1.6 cells were still inhibited by 20 nM charybdotoxin (Fig. 4A) showing that genetic deficiency in p56lck kinase did not alter the channels' properties. Finally, reconstitution of p56lck into JCaM 1.6 cells partially restored the inhibitory effect of anti-human Fas (Fig. 4B).Fig. 4Herbimycin A or genetic deficiency of p56lck tyrosine kinase prevents Fas-induced inhibition of n-type K+ channels. A, whole-cell currents elicited by depolarizing +40 mV pulses at 30-s intervals from −70 mV holding potential in control conditions (a), after 17 min of incubation with 200 ng/ml anti-Fas antibody (b), and after perfusion with 20 nM charybdotoxin (c) in a JCaM 1.6 cell. B, alterations of peak currents following addition of 200 ng/ml anti-Fas antibody in percent of control peak current in Jurkat cells without (n = 8) and with pretreatment with 10 µM herbimycin A (n = 4) for 6 h, as well as in JCaM 1.6 without (n = 5) and with (n = 5) p56lck reconstitution after 16 ± 2, 15 ± 2, 17 ± 2, and 22 ± 4 min of incubation, respectively. Data are expressed as arithmetic means ± S.E. Differences are statistically significant (t test for independent samples, one-tailed) between Jurkat (<1108>) and Jurkat + herbimycin A () (p < 0.0007), between Jurkat and JCaM 1.6 (▧) (p < 0.0007), and between JCaM 1.6 and JCaM 1.6 + p56lck cells (bricked column) (p < 0.0372).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe data summarized above show that activation of the Fas receptor elicits rapid tyrosine phosphorylation of the Kv1.3 K+ channel protein and inhibition of the n-type K+ channel. Furthermore, we demonstrate that the p56lck tyrosine kinase deficiency and tyrosine kinase inhibition almost abolish the inhibitory effect of Fas on the channel. Even though the lack of p56lck kinase activity completely prevented tyrosine phosphorylation as revealed by phosphorylation analysis, the Fas-induced channel inhibition was not completely abolished. These results suggest that direct tyrosine phosphorylation of the channel by p56lck tyrosine kinase and/or by a downstream tyrosine kinase accounts for suppression of channel activity; however, an additional mechanism may operate as well to inhibit the n-type channel following Fas stimulation.The shaker-family member Kv1.2 channels, that show high a degree of homology with Kv1.3 (15Pongs O. Physiol. Rev. 1992; 72: S69-S88Crossref PubMed Google Scholar), are known to be down-regulated by tyrosine phosphorylation (16Huang X.Y. Morielly A.D. Peralta E.G. Cell. 1993; 75: 1145-1156Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 17Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Sclessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). Similarly, phosphorylation of the n-type K+ channels in Jurkat cells by protein kinases A and C result in suppression of the current (6Payet M.D. Dupuis G. J. Biol. Chem. 1992; 267: 18270-18273Abstract Full Text PDF PubMed Google Scholar).The n-type voltage-gated K+ channels have been shown to be responsible for the maintenance of the resting membrane potential in lymphocytes, since their inhibition by the specific inhibitors margatoxin and noxiustoxin (18Garcia-Calvo M. Leonard R.J. Novick J. Stevens S.P. Schmalhofer W. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1993; 268: 18866-18874Abstract Full Text PDF PubMed Google Scholar, 19Bednarek M.A. Bugianesi R.M. Leonard R.J. Felix J.P. Biochem. Biophys. Res. Commun. 1994; 198: 619-625Crossref PubMed Scopus (42) Google Scholar) leads to a marked depolarization (20Leonard R.J. Garcia M.L. Slaughter R.S. Reuben J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10094-10098Crossref PubMed Scopus (265) Google Scholar). Changes in membrane potential seems to be an early and important event upon mitogenic stimulation of lymphocytes (21Tsien R.Y. Pozzan T. Rink T.J. Nature. 1982; 295: 68-71Crossref PubMed Scopus (768) Google Scholar). T cell activation selectively increases the number of n-type K+ channels (22De Coursey T.E. Chandy K.G. Gupta S. Cahalan M.D. J. Neuroimmunol. 1985; 10: 71-95Abstract Full Text PDF PubMed Scopus (149) Google Scholar), which might in part lead to the hyperpolarization observed by Tsien et al. (21Tsien R.Y. Pozzan T. Rink T.J. Nature. 1982; 295: 68-71Crossref PubMed Scopus (768) Google Scholar). Margatoxin- and noxiustoxin-induced depolarization blocks proliferation and lymphokine production in T lymphocytes by affecting the Ca2+-dependent pathways involved in proliferation (23Lin C.S. Boltz R.C. Blake J.T. Nguyen M. Talento A. Fischer P.A. Springer M.S. Sigal N.H. Slaughter R.S. Garcia M.L. Kaczorowski G.J. Koo G.C. J. Exp. Med. 1993; 177: 637-645Crossref PubMed Scopus (239) Google Scholar).The n-type K+ channels are involved in the regulatory volume decrease (24Grinstein S. Smith J.D. J. Gen. Physiol. 1990; 95: 97-103Crossref PubMed Scopus (89) Google Scholar, 25Deutsch C. Chen L.Q. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10036-10040Crossref PubMed Scopus (154) Google Scholar) as well. Their activation leads to shrinkage. Volume regulation might be prevented by inhibition of the n-type K+ channels in lymphocytes.We suggest that changes in membrane potential might be important in apoptosis. This idea is supported by our observation in which the rate of apoptotic death, measured by DNA degradation, is slightly but significantly increased (10 ± 3%) when cells were incubated with anti-human Fas antibody and margatoxin together, compared to the cell death in the case of incubation with anti-human Fas alone. Since n-type K+ channels have been implied in volume regulatory decrease as well, their inhibition might regulate cell volume, which in turn might interfere with the apoptotic pathway. The exact mechanism by which inhibition of n-type K+ channels might influence apoptosis and the signal transduction pathway however is still unknown. Furthermore, Fas triggering might regulate other ion channels as well, a possibility currently being investigated in our laboratory. INTRODUCTIONThe programmed cell death is a process characterized by DNA fragmentation and apoptotic cell morphology. Apoptosis can be triggered by a variety of extrinsic and intrinsic signals, including ligation of APO-1/Fas (CD95) antigen or purified Fas ligand (1Suda T. Nagata S. J. Exp. Med. 1994; 179: 873-879Crossref PubMed Scopus (499) Google Scholar, 2Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3965) Google Scholar). One of the crucial events in the early phase of Fas-induced apoptosis is the tyrosine phosphorylation of multiple cellular proteins (3Eischen C.M. Dick C.J. Leibson P.J. J. Immunol. 1994; 153: 1947-1954PubMed Google Scholar). Incubation of lymphocytes with herbimycin A, an inhibitor of Src-like tyrosine kinases prevents Fas-induced apoptosis (3Eischen C.M. Dick C.J. Leibson P.J. J. Immunol. 1994; 153: 1947-1954PubMed Google Scholar).Thus far, nothing is known about the possible involvement of ion channels in apoptotic cell death. The present study has been performed to test for an influence of Fas stimulation on ion channel activity. As a result, Fas indeed led to a striking inhibition of n-type K+ channels, the most abundant K+ channels in lymphocytes. To further elucidate the mechanism of this inhibition, we looked for phosphorylation of the channel protein following Fas stimulation. Triggering of the Fas receptor was indeed followed by tyrosine phosphorylation of the channel. In order to evaluate the significance of the phosphorylation for channel inhibition, we have performed studies examining the effect of Fas on n-type K+ channel activity in cells deficient of tyrosine kinase and in the presence of tyrosine kinase inhibitor.In the present study we show that the n-type K+ channels are inhibited upon Fas stimulation of Jurkat T lymphocytes and that the suppression of current is correlated with tyrosine phosphorylation of the channel protein by the p56lck tyrosine kinase.