Lymphocyte-specific inducible expression of potassium channel beta subunits

Journal of Neuroimmunology 77 Ž1997. 8–16

Lymphocyte-specific inducible expression of potassium channel beta subunits Michael V. Autieri a , Stanley M. Belkowski a , Cristian S. Constantinescu b, Jeffrey A. Cohen c , Michael B. Prystowsky a,) a

c

Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park AÕenue, Bronx, NY 10461, USA b Department of Neurology, UniÕersity of PennsylÕania School of Medicine, Philadelphia, PA, USA Mellen Center for Multiple Sclerosis Treatment and Research, CleÕeland Clinic Foundation U-10, 9500 Euclid AÕe., CleÕeland, OH 44195, USA Received 30 August 1996; revised 28 January 1997; accepted 31 January 1997

Abstract Many studies have shown that voltage-gated potassium ŽKv. channel activity is essential for T-lymphocyte proliferation. The IL-2-inducible neuroimmune gene, I2rf5 is the mouse homologue of the rat Kvb 2 subunit. In this study we show that in addition to constitutive expression in adult murine brain, expression of Kv channel subunits b 1.1 and b 2.1 is inducible in a cloned T-helper cell line stimulated with IL-2 and in normal murine splenocytes stimulated with Con A or LPS. This expression pattern appears to be lymphocyte specific, because stimulated fibroblasts and vascular smooth muscle cells do not express Kvb channel subunit mRNA. These observations suggest that Kvb subunit expression is tissue specific and inducible in stimulated lymphocytes. Because Kvb subunits modulate Kq channel activity, the inducible and variable expression of these subunits in lymphocytes may represent an additional regulatory mechanism for lymphocyte proliferation. Keywords: T-cells; Potassium channels; B subunits; IL-2; Lymphycyte activation

1. Introduction Voltage-gated potassium ŽKv. channels are necessary for maintenance of membrane potential, signal transduction and current generation in many cell systems. Kv channels consist of two subunits: Ž1. a pore forming a subunit and Ž2. an intracytoplasmic b subunit ŽAldrich, 1994.. The family of Kva subunit genes Ž shaker, shakerrelated. consists of multiple loci on at least 5 mouse chromosomes ŽAdams et al., 1992; Schwartz et al., 1988.. Similar to Naq and Ca2q channels, the functional characteristics of pore-forming a subunits of these voltage-gated ion channels are modified by a number of accessory proteins ŽAldrich, 1994.. Recently, five members of a new family of Kvb subunits have been cloned, four of which interact and modify Kva subunit function ŽAdams et al., 1992; Scott et al., 1994; Majumder et al., 1995; Rettig et al., 1994; England et al., 1995; Heinemann et al., 1995..

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Corresponding author. Tel.: q1-718-4302827; fax: q1-718-4308541.

There are two major types of Kv channels: Ž1. a slowly inactivating delayed-rectifier type and Ž2. a rapidly inactivating A-type Kv channel. Oligomers of the a subunit alone can form the delayed rectifier; particular combinations of a and b subunits result in hetero-oligomers with 1:1 stoichiometry and in the conversion to rapidly inactivating Kv channels ŽScott et al., 1994; Majumder et al., 1995; Rettig et al., 1994; Morales et al., 1995.. The cloning and expression of multiple highly conserved, tissue specific and developmentally regulated a subunit genes suggests the existence of tissue specific and developmentally regulated b subunits as well. Several observations indicate that potassium channel function is important in the triggering andror support of mitogen-induced T-lymphocyte proliferation: Ž1. activated lymphocytes have increased K q conductance when compared to quiescent lymphocytes ŽLee et al., 1986; DeCoursey et al., 1984., Ž2. lymphocyte activation is inhibited by membrane depolarization ŽGelfand et al., 1987., Ž3. lectin mitogens cause an immediate shift in the relative K q conductance ŽChandy et al., 1984; Cahalan et al.,

0165-5728r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 7 . 0 0 0 5 0 - 7

M.V. Autieri et al.r Journal of Neuroimmunology 77 (1997) 8–16

1985. and Ž4. pharmacological agents that block K q conductance inhibit mitogen-driven lymphocyte proliferation ŽLee et al., 1988.. While both B- and T-lymphocytes express functional Kv channels during activation, the subunits required for Kv channel activity Ž a subunit with or without b subunit. and the regulation of expression of each subtype in lymphocytes is unknown. A major effort in our laboratory has been directed towards elucidating molecular mechanisms regulating lymphocyte proliferation. While identifying genes expressed during IL-2-driven T-cell proliferation, we previously isolated and characterized a sequence designated F5 which was shown to be expressed only in proliferating T-lymphocytes and mature, post mitotic neurons ŽSabath et al., 1990; Cohen et al., 1992.. Recent nucleic acid sequence analysis has revealed that this sequence is identical to a rat Kv channel sequence, b 2.1 ŽScott et al., 1994. which shares 73% amino acid homology with human Kvb 1.2 and 74% amino acid homology with rat Kvb 1.1. In these studies we examine the distinct temporal expression of each beta subunit in stimulated lymphoid cells and show that Kvb 1.1 and b 2.1, but not b 1.2 are inducible in activated lymphocytes. No Kvb subunit mRNA is detectable in cytokine stimulated cells of other lineages tested, suggesting specialized roles for Kvb 1.1 and Kvb 2.1 in lymphocyte proliferation.

2. Materials and methods 2.1. Cell culture The derivation and maintenance of L2 cells, an alloreactive mouse helper T-cell clone of B6 origin ŽH-2 b ., has been described elsewhere ŽSabath et al., 1990; Glasebrook et al., 1981.. Briefly, cells were maintained in Dulbecco’s Modified Eagle Medium with 4.5 mgrml glucose ŽWhittaker Bioproducts, Walkersville, MD. supplemented with 100 Urml penicillin, 100 mgrml streptomycin, 4 mM glutamine, 50 mM 2-mercaptoethanol, 10% heat-inactivated fetal calf serum ŽFCS, HyClone Laboratories, Logan, UT. - DMEMrCM. For expansion, L2 cells at a concentration of 1 = 10 6 cellsrml were co-cultured with irradiated allogeneic CBArJ spleen cells ŽH-2 d . at a concentration of 4 = 10 7 cellsrml in 25 ml DMEMrCM containing 100 Urml of IL-2. Human recombinant IL-2 was a gift of Chiron ŽEmeryville, CA. ŽRosenberg et al., 1984.. This substance was 98% pure and contained 0.01 ng of endotoxinr3.6= 10 6 units of IL-2; specific activity was evaluated by the manufacturer. Murine splenocytes were isolated by Ficoll-Hypaque density-gradient centrifugation and cultured in DMEMrCM containing 5 m grml concanavalin A ŽCon A.. BALBrc 3T3 and NIH3T3 mouse fibroblasts were maintained in DMEMrCM plus 100 Urml penicillin, 100 mgrml streptomycin and 10%

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FCS. Rapamycin Ž20 ngrml., purchased from LC laboratories ŽWoburn, MA. was added to cultures at the same time as IL-2 ŽFeuerstein et al., 1995.. Rat vascular smooth muscle cells ŽVSMC. from carotid artery explants were grown and subcultured in growth medium ŽDMEM, 10% FCS, L-glutamine.. Cells from passage 5–9 were used in the described studies. Pre-confluent VSMCs were serum starved for 48 h in Dulbecco’s minimum essential media, then exposed to 10% fetal calf serum, for the times indicated. 2.2. RNA isolation and northern blot analysis For each time point studied, cells from culture were isolated and total RNA obtained according to standard methods as described previously ŽAutieri et al., 1995.. Equal amounts of RNA were loaded and separated on a 1.3% agaroserformaldehyde gel, transferred to nitrocellulose and hybridized in buffer containing 0.25 M NaCl, 1% sodium dodecyl sulfate, 50% formamide, 2 = Denhardt’s solution, 25 m g denatured salmon sperm DNA, 5% dextran sulfate at 428C overnight with the indicated probe. Blots were washed under high stringency Ž0.2 = sodium citrate, 0.1% sodium dodecyl sulfate, 658C., and exposed to film for 6–48 h at y808C. All probes were w a32 Px-labeled by the random priming method or w g32 Px labeled by polynucleotide kinase ŽBoehringer Mannheim, Indianapolis, IN. Žall isotopes were from Amersham, Arlington Heights, IL.. The same filter was stripped and subsequently hybridized with the various DNA probes. Inserts of the following constructs were used as probes: pGEM F5.1, an 853-bp BamHI fragment corresponding to 81 bp of 5X untranslated sequence and the first 772 bp of the protein coding sequence of a mouse brain ŽKvb 2.1. cDNA Ž14.; pUC25, mouse T-cell receptor b chain ŽHashimoto and Blank, 1990.; pH5.3, mouse proliferating cell nuclear antigen ŽShipman et al., 1988.; Kvb-specific probes were made by synthesis of 45 bp antisense oligonucleotides corresponding to areas unique to each published Kvb cDNA sequence as follows: rat Kvb 1.1 nt 385-nt 430, rat Kvb 2.1, nt 450-nt 495 and human Kvb 1.2, nt 69-nt 114 ŽFig. 1B.. This region of the human b 1.2 cDNA is identical in 44 of 45 bp to the recently cloned ferret b 1.2 cDNA ŽMorales et al., 1995.. The glyceraldehyde-3-phosphate dehydrogenase ŽG3PDH. probe was generated from PCR amplimers ŽClonetech, Palo Alto, CA.. Relative intensities of hybridization signals were obtained by densiometric scanning ŽRFLP-Scan Software, Scanalytics. of autoradiograms exposed within the linear range of the film ŽKodak X-OMAT.. 2.3. Western blot analysis Rabbit antiserum corresponding to the 18 C-terminal residues of the deduced F5 ŽKvb 2.1. protein was described previously ŽArai and Cohen, 1993.. Briefly, L2

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Fig. 1. ŽA. Alignment of predicted amino acid sequence of the cDNA for the neuroimmune gene I2rf5 and Kv channel beta subunits 1.1, 2.1 and 1.2 ŽGenBank accession numbers U31908, X70661, X70662 and U16953, respectively.. Asterisks indicate residues common to all three genes and filled circles indicate identity between b 1.1 and b 1.2. ŽB. Schematic representation of Kvb subunit cDNA chosen for oligonucleotide probes. Lines represent X the 5 UTR of each cDNA, blocked areas represent the coding sequences and the underscore represents the oligonucleotide probes. The nucleotides for each of the antisense probes are shown. The b 1.1 probe represents amino acids 19–33 of the b 1.1 amino acid sequence ŽA.. The b 1.2 probe represents amino X acids 1–16 of the b 1.2 sequence ŽA.. The b 2.1 probe represents a portion of the 5 UTR of the b 2.1 sequence.

M.V. Autieri et al.r Journal of Neuroimmunology 77 (1997) 8–16

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cells were cultured and treated with or without IL-2 as described above and adult mouse brain ŽBalbrc. was homogenized in RIPA buffer ŽPBS containing 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% SDS, 0.2 Urml aprotinin, 2 mM EDTA, 10 mM sodium fluoride, 400 m M sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM PMSF.. Proteins were size-fractionated on a 10% polyacrylamide gel by the method of Laemmli ŽLaemmli, 1970. and transferred to polyvinylidene difluoride membranes ŽImmobilon-P, Millipore. by the method of Towbin ŽTowbin et al., 1979.. Membranes were blocked in 100 mM TBS containing 10% nonfat dried milk and 0.05% Tween 20. A saturating concentration of affinity purified anti-Kvb 2 antibody was recognized by an alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody ŽBoehringer-Mannheim.. Membranes were washed extensively in 100 mM TBS containing 3% nonfat dried milk and 0.05% Tween 20. Immunoreactivity was visualized by an alkaline phosphatase-catalyzed color reaction.

3. Results Recent nucleic acid sequence analysis has revealed that the sequence originally identified as F5, cloned from a murine IL-2-induced T helper cell library is identical to the b chain ŽKvb 2.1. of voltage-gated potassium channels; a sequence comparison of the three known Kvb chains including Kvb 1.1 Žrat. ŽRettig et al., 1994., Kvb 2.1 Žrat. ŽScott et al., 1994. and Kvb 1.2 Žferret and human. ŽMajumder et al., 1995; Morales et al., 1995. is shown in Fig. 1A. Mouse Kvb 2.1 ŽF5. cDNA, containing a large 3X UTR is 3586 bp in length and likely contains the complete 3X end of the transcript, whereas rat Kvb 2.1, at 1698 bp, appears to encompass the full length 5X terminus ŽScott et al., 1994.. Kvb 2.1 has 74% amino acid identity to Kvb 1.1. Kvb 2.1 is 73% identical to a third isoform, Kvb 1.2, cloned from a ferret atrial library ŽMorales et al., 1995.. The three subunits are highly conserved and differ almost exclusively in the length and sequence of their N-terminal regions, with mouse Kvb 2.1 being 85% identical to b 1.1 and b 1.2 in a 329 amino acid C-terminal span. The b 1.2 subunit is 9 amino acids longer than b 1.1 and 39 amino acids longer than b 2.1. It has been suggested that the N-terminal region of b 1.1 contains a single putative ball peptide sequence and based on alignment relative to a conserved cysteine residue, b 1.2 would contain two ball-peptide-like sequences, whereas b 2.1 would contain none ŽRettig et al., 1994; Rehm and Lazdunski, 1988.. 3.1. IL-2-stimulated KÕb chain expression Using a cDNA probe corresponding to the entire mouse Kvb 2.1 protein coding region and a portion of 3X UTR, previous results indicate that Kvb expression is induced in

Fig. 2. Expression of Kvb subunit mRNA in IL2-stimulated L2 cells. ŽA. Total RNA was isolated from the murine T-helper cell L2 at varying times post-IL2 Ž100 Urml. stimulation and analyzed by northern blot analysis using oligonucleotide probes specific for rat Kvb 1.1, Kvb 2.1 and ferret Kvb 1.2. RNA was isolated from cells at the following time points: Ž1. quiescent; Ž2. 6 h; Ž3. 12 h; Ž4. 24 h; Ž5. 36 h and Ž6. 72 h. RNA loading and degradation was assessed by staining with ethidium bromide and review of 18s and 28s rRNA bands. 12 m g of total RNA was analyzed in each lane and the same filter was stripped and sequentially hybridized with the respective probes as described in Section 2. A proliferating cell nuclear antigen ŽPCNA. probe was used as an activation control, and a T-cell receptor b chain ŽTb . probe was used to assess relative levels of RNA accumulation. Autoradiographic exposure times were 48 h for all probes, with the exception for Kvb 1.2, which was exposed for 6 days to enhance signal detection. ŽB. Scanning densitometric analysis of northern blots of Kvb subunit mRNA expression in stimulated L2 cells. Y-axis corresponds to Kvb subunit expression and are normalized for TCR b chain expression; X-axis corresponds to time-post IL2-stimulation.

T-lymphocytes by IL-2 and expression is inhibited by cycloheximide ŽSabath et al., 1990; Cohen et al., 1992.. These studies could not distinguish specific Kvb subunit expression ŽSabath et al., 1990.. To examine the temporal expression of Kvb 1.1, b 2.1 and b 1.2 during IL-2-driven T-cell proliferation, total RNA was prepared from resting and IL-2-stimulated L2 cells 6, 12, 24, 36 and 72 h after IL-2 addition and probed using an end-labeled antisense oligonucleotides corresponding to 5X sequence unique to each Kvb subunit. Fig. 2A shows both Kvb 1.1 and b 2.1

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subunits are inducible in IL-2-stimulated T-lymphocytes, but with differing kinetics. For Kvb 1.1, a small amount of message was detectable in resting cells; expression rapidly increased at 6 h to near maximal levels and was sustained throughout the 72 h time course. In contrast, no Kvb 2.1, message was detected in resting T-cells; b 2.1 mRNA was first observed at 6 h after addition of IL-2, increased to

Fig. 4. Expression of Kvb 1.1 and Kvb 2.1 RNA accumulation are unaffected by rapamycin. ŽA. Total RNA was isolated from the murine T-helper cell L2 at Ž1. 0 h, Ž2. 24 h post IL2 stimulation, Ž3. 24 h post IL2 plus 20 ngrml rapamycin and Ž4. 24 h plus rapamycin alone and hybridized with probes specific for rat Kvb 1.1 and Kvb 2.1. 15 m g of total RNA were analyzed in each lane and the same filter was stripped and sequentially hybridized with the respective probes as described in Section 2. A proliferating cell nuclear antigen ŽPCNA. probe was used as an activation and rapamycin sensitivity control. Autoradiographic exposure times were 48 h for all probes. 3 H-thymidine incorporation was as follows: 0 h s 3,930"2,700; 24 h plus IL2 s 40,240"3,450; 24 h plus IL2 and rapamycins 5,750"1,900. ŽB. Scanning densitometric analysis of northern blots of Kvb subunit mRNA expression. Y-axis corresponds to Kvb subunit expression; various treatments are given on the X-axis. Fig. 3. Kvb subunit RNA accumulation in stimulated mouse splenocytes. ŽA. Total RNA was isolated from freshly dissociated adult Balbrc mouse splenocytes at varying times after stimulation with Con A Ž5 m grml. and analyzed by northern blot analysis using oligonucleotide probes specific for rat Kvb 1.1, Kvb 2.1 and ferret Kvb 1.2. RNA was isolated from the cells at the following time points: Ž1. freshly isolated; Ž2. 6 h; Ž3. 15 h; Ž4. 24 h; Ž5. 48 h; Ž6. 72 h and Ž7. 120 h. RNA loading and degradation was assessed by staining with ethidium bromide and review of 18s and 28s rRNA bands. 12 m g of total RNA were analyzed in each lane and the same filter was stripped and sequentially hybridized with the respective probes as described in Section 2. Lane 8 is total RNA from mouse splenocytes stimulated with lipopolysaccharide ŽLPS. for 24 h and lane 9 is 8 m g total RNA isolated from adult mouse ŽBalbrc. brain. A proliferating cell nuclear antigen ŽPCNA. probe was used as an activation control. Autoradiographic exposure times were 48 h for all probes, with the exception for Kvb 1.2, which was exposed for 6 days to enhance signal detection. ŽB. Scanning densitometric analysis of northern blots of Kvb subunit mRNA expression in Con A-stimulated splenocytes. Y-axis corresponds to Kvb subunit expression; X-axis corresponds to time-post Con A-stimulation.

three fold this amount at 12 h, reached near maximal levels at 24 h and was sustained at this level through 72 h ŽFig. 2B.. The initial time course induction of b 2.1 RNA accumulation is similar to that observed for proliferating cell nuclear antigen ŽPCNA., an IL-2-induced gene required for proliferation ŽShipman et al., 1988., but PCNA RNA levels decrease later in the time course when cells stop proliferating. b 1.2 mRNA was not detected. 3.2. KÕb chain expression in mitogen-stimulated splenocytes To determine if beta subunit expression is inducible in lymphocytes freshly obtained from animals, mouse splenocytes were stimulated with concanavalin A ŽCon A. and

M.V. Autieri et al.r Journal of Neuroimmunology 77 (1997) 8–16

RNA levels were measured. As shown in Fig. 3A, similar to that observed in IL-2-stimulated L2 cells, expression of Kvb subunits is inducible in Con A-stimulated splenocytes. Interestingly, in contrast to IL-2-stimulated cloned T-cells, identical expression patterns were observed for Kvb 1.1 and b 2.1 subunits, with a low basal level of mRNA detectable in resting spleen cells, a marked increase within 15 h of stimulation peaking by 72 h. By 120 h after stimulation, the level of Kvb 1.1 and b 2.1 mRNA decreased substantially, although not quite back to baseline ŽFig. 3B.. This expression pattern is quite similar to that observed for PCNA. Kvb 1.2 mRNA was not detected. To determine if Kvb 1.1 and b 2.1 inducible expression is a T-cell specific phenomenon, mouse splenocytes were stimulated with the B-lymphocyte mitogen lipopolysaccharide ŽLPS. for 24 h and b subunit RNA levels were measured. Similar to T-lymphocytes, both b 1.1 and b 2.1 mRNA were expressed to a similar degree in LPS-stimulated splenocytes ŽFig. 3A, lane 8.. As in stimulated T-cells, Kvb 1.2 message was not detectable. Since Kvb 1.1 and b 2.1 are also expressed in the brain ŽScott et al., 1994., total RNA from adult mouse brain was analyzed for b subunit expression. Kvb 1.1 and b 2.1, but not b 1.2 or PCNA are expressed in brain ŽFig. 3A, lane 9.. The observation that Kvb 1.1 and Kvb 2.1 are induced in proliferating lymphocytes and yet are constitutively expressed in non-proliferating tissue such as adult brain, suggests a special requirement for this subunit in regulating potassium channel function. 3.3. Cell cycle linked b subunit expression The immunosuppressant drug rapamycin blocks T-cell proliferation by inhibiting molecular events in the IL-2 receptor pathway, including PCNA expression ŽFeuerstein et al., 1995., resulting in growth arrest in the G1 phase of the cell cycle Žfor review, Kunz and Hall, 1993.. Because use of rapamycin represents a useful tool to dissect T-cell proliferative events ŽBrown et al., 1994., we examined

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Fig. 6. Kvb subunit RNA accumulation in stimulated rat vascular smooth muscle cells ŽVSMC.. Total RNA was isolated from rat VSMC at varying times following 10% fetal calf serum stimulation ŽFCS. and analyzed by northern blot analysis using oligonucleotide probes specific for rat Kvb 1.1, Kvb 2.1 and ferret Kvb 1.2. RNA was isolated from the cells at the following time points: Ž1. serum-starved 48 h; Ž2. 12 h; Ž3. 24 h; Ž4. 48 h and Ž5. 120 h. RNA loading and degradation was assessed by staining with ethidium bromide and review of 18s and 28s rRNA bands. 20 m g of total RNA was analyzed in each lane and the same filter was stripped and sequentially hybridized with a cDNA probe corresponding to the entire protein coding region of Kvb 2 ŽF5. cDNA, as described in Section 2. A proliferating cell nuclear antigen ŽPCNA. probe was used as a proliferation control, and a glyceraldehyde-3-phosphate dehydrogenase ŽG3PDH. probe was used to assess relative levels of RNA accumulation. Autoradiographic exposure times were 6 days for all probes to enhance signal detection.

IL-2-inducible expression of Kvb subunits in the presence and absence of this compound. Fig. 4 shows that in contrast to PCNA, expression of both b 1.1 and b 2.1 subunit RNA are insensitive to rapamycin, suggesting that unlike PCNA, expression of these genes involves a rapamycin-insensitive activation pathway. 3.4. b subunit protein expression Antiserum specific for the C-terminal segment octadecapeptide of Kvb 2.1 has been used previously to detect b subunit expression in brain ŽArai and Cohen, 1993.. This antiserum should recognize a region common to all Kvb subunit proteins. Similar to RNA levels, little to no beta subunit protein is detected in resting L2 cells. In IL-2stimulated T-lymphocytes, two proteins are detected, including a strong band at 42 kDa and a fainter band at 39 kDa ŽFig. 5.. These sizes are identical to Kvb 1.1 and b 2.1 channel subunits purified from bovine brain by an a dendrotoxin affinity purification scheme ŽScott et al., 1994. and indicate that expression of these beta subunits is not limited to neuronal tissue and that expression is inducible in activated lymphocytes. 3.5. Tissue-specific inducible b subunit expression

Fig. 5. Expression of Kvb protein in L2 cells stimulated with IL2. Cell extracts were analyzed for Kvb protein levels by western analysis as described in Section 2. Lane 1, unstimulated L2 cells; lane 2, L2 cells stimulated for 24 h with IL2 Ž100 Urml. and lane 3, extract from whole adult mouse brain.

To determine if potassium channel beta subunit expression is a general response to cellular proliferation, other inducible cell systems were examined. Rat vascular smooth muscle cells ŽVSMC. provide a good model to study cellular proliferation because of their ability to dedifferentiate and reenter the cell cycle upon mitogen stimulation.

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Rat VSMC were grown to just under confluency, serum starved 48 h to quiescence and stimulated with 10% FCS for varying lengths of time. Activation of these cells was confirmed by PCNA expression. In contrast to mouse lymphocytes, no Kvb 1.1, b 2.1, or b 1.2 subunit expression was detected up to 72 h in rat VSMC, ŽFig. 6.. A marked increase in PCNA expression was observed 16 h after readdition of FCS, confirming that these cells were activated. In additional experiments, no beta subunit induction was observed in NIH3T3 fibroblasts or NIH3T3 cells stimulated to proliferate by re-addition of 10% FCS to serum-starved cultures, LPS-stimulated mouse macrophages, or in regenerating liver following partial hepatectomy Ždata not shown.. Taken together, these data indicate that inducible expression of potassium channel beta subunits is not a general property of proliferating cells in culture and appears to be restricted to cells within lymphocyte lineages.

4. Discussion Like many other voltage-gated ion channels, Kq channels are composed of subunits that are presumed to play regulatory roles in channel activity. The a subunit, consisting of transmembrane domains forms the intact Kq channel but when expressed alone, does not fully reproduce all properties of native voltage-gated channels ŽAldrich, 1994; Rettig et al., 1994.. In recent co-transfection experiments, it has been shown that b 1.1 increases the inactivation of the Kv1.1 ŽRCK1. a subunit 100 fold, but b 1.2 had no effect on Kv1.1. However, b 1.2 increases the rate of inactivation of Kv1.4 ŽRCK4. or Shaker 4–7 fold. The Kvb 2.1, subunit did not affect the properties of RCK1 or RCK4. Thus, specific combinations of particular Kva and Kvb subunits determine the conductive properties of channels and, likely, cellular function. An important role for Kq ion flux in lymphocyte activation has been demonstrated, as inhibition of current by Kq channel blockers inhibits IL-2-stimulated volume increases, protein synthesis and cell cycle progression ŽLee et al., 1986; Chandy et al., 1984.. IL-2-stimulated lymphocytes undergo large changes in volume during the proliferative process. The ability of T-cells to regulate volume in response to hypotonicity is proportional to the magnitude of Kv channel conductance ŽLee et al., 1988.. Thus, it appears that at least one important physiologic role for Kv channels in lymphocyte proliferation appears to be volume regulation ŽDeutsch and Chen, 1993.. The neuroimmune gene, I2rf5, cloned originally as a cDNA from an IL-2-stimulated T-helper cell library, is identical with the Kvb 2.1 subunit, suggesting a role for this subunit in lymphocyte proliferation ŽFig. 1.. Kvb 2.1 is 85% identical with Kvb 1.1 and b 1.2 over 329 C-terminal amino acids, but shares no identity with Kvb 1.1 or b 1.2 over 79 N-terminal amino acids. The sharp transition

to a non-conserved region from a highly conserved Cterminal region between Kvb 1.1, b 2.1, and b 1.2 suggests that functional differences in Kvb subunits may be related to N-terminal sequence differences. In the nervous system, the b 1.1 subunit modulates Kv properties as a function of an N-terminal cysteine residue redox state ŽRettig et al., 1994.. Based on alignment relative to a conserved cysteine residue, it has been determined that the N-terminal region of b 1.1 contains a single putative ball peptide sequence and b 1.2 would contain two ball-peptide-like sequences, whereas b 2.1 would contain none ŽRettig et al., 1994; Rehm and Lazdunski, 1988.. Because Kvb 2.1 has not been shown to alter Kv channel functionality from a delayed-rectifier type to an A type Kv channel, it is presumed that the functional differences observed between the Kvb subunits are likely attributable to the sequence differences in the N-terminal domain. Variable expression of Kvb subunits with or without ball peptide sequence may act as an additional regulatory mechanism in Kq channel activity by setting the rate of K channel inactivation. The cloning and expression of multiple highly conserved, tissue specific, and developmentally regulated Kva subunit genes suggests the existence of tissue specific and developmentally regulated Kvb subunits as well. Fig. 2 shows the temporal expression pattern of Kvb 1.1, and b 2.1 in IL-2-stimulated L2 cells. These cells have been shown previously to have potassium channel conductance characteristics very similar to that found in human peripheral blood lymphocytes and a murine cytotoxic T-cell line ŽLee et al., 1986, 1988; Deutsch and Chen, 1993; Matteson and Deutsch, 1984.. Although it is clear that both Kvb 1.1 and b 2.1 are IL-2-inducible in equal magnitude, the time courses of expression are not identical. Kvb 1.1 demonstrates a basal mRNA level in resting T-cells and induction occurs much more rapidly than Kvb 2.1. The slower induction of Kvb 2.1 may act as a modulatory mechanism in the later stages of lymphocyte activation by converting a greater proportion of K q channels to the slowly inactivating type. Kvb 1.2, isolated from a ferret cDNA library, is not expressed in L2 cells. This lack of expression suggests that Kvb 1.2 may be tissue-restricted. Our findings of Kvb subunit expression in IL-2-stimulated L2 cells were extended to freshly isolated lymphocytes. Con A-stimulation confirms that both Kvb 1.1 and Kvb 2.1 are expressed in freshly isolated T-cells. The more subtle finding of a difference in the time course of RNA accumulation for Kvb 1.1 and Kvb 2.1 was not observed with Con A-stimulation and may be specific for IL-2-induced expression. In B-lymphocytes, induction of potassium channel function depends on the pathway of stimulation ŽPartiseti et al., 1993.. The B-lymphocyte mitogen, LPS, also induces Kvb 1.1 and Kvb 2.1 expression to a similar degree. Because Kvb and PCNA RNA accumulation are increased by the same stimuli in T-cells with similar time

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courses, there was a possibility that these genes were coordinately regulated. Since rapamycin shows a rather selective inhibition of PCNA expression ŽFeuerstein et al., 1995., the ability of rapamycin to block Kvb expression appeared to be a good diagnostic for coordinate gene expression. Rapamycin did not inhibit Kvb RNA accumulation indicating that the activation pathway responsible for Kvb subunit expression differs from the one responsible for PCNA expression. Several functional studies have shown that the number of voltage-gated or calcium gated Kq channels increase during activation of various cell types with mitogens ŽGelfand et al., 1987; Chandy et al., 1984; Lee et al., 1988; Matteson and Deutsch, 1984; Partiseti et al., 1993. leading to the suggestion that induction of Kq channel expression is the consequence of a stereotyped cellular response to entry into the cell cycle ŽPartiseti et al., 1993.. Fig. 6 shows that Kvb subunits are not expressed in proliferating rat VSMC. Similar results were obtained in NIH3T3 cells stimulated to proliferate, LPS-stimulated mouse macrophages and in regenerating hepatocytes, indicating that inducible expression of Kvb subunits is not a general consequence of cellular proliferation, but is restricted to lymphocytes. Expression of particular Kva channels is regulated both in a tissue-specific and developmentally specific manner. Kv1.5, a shaker-like delayed rectifier channel is tissuespecifically and developmentally expressed in heart, skeletal muscle and brain ŽBeckh and Pongs, 1990; Mori et al., 1993; Matsubara et al., 1991.. The 5X regulatory region of this gene contains a cAMP regulatory element ŽCRE. and transcription is increased by cAMP-dependent protein kinase ŽPKA. activation ŽBosma et al., 1993; McNicholas et al., 1994.. Investigation into the 5X regulatory region of Kvb subunits should reveal if particular combinations of Kva and Kvb subunits are coordinately regulated. The immune and nervous systems are recognized to be intimately involved in bi-directional communication and share many common cell surface receptors and ligands ŽKelley, 1989; Bost, 1988; Aird et al., 1993.. There is also a class of genes which is predominantly or exclusively expressed in neural and immune systems ŽFarrar et al., 1989; Parnes and Hunkapiller, 1989.. The neuronal and lymphocyte-restricted expression suggests that Kvb 2.1 may be a member of this class of proteins. The differential expression of Kv beta subunits in lymphocytes may represent an additional mechanism of regulation of membrane potential and induction of mitogen-induced proliferation and underscores the importance of further investigations into the function and expression of Kq channel subunits in lymphocytes. Acknowledgements M.B.P. is the recipient of an American Cancer Society Faculty Research Award. Supported in part by grant

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MH51327 from the NIMH and Yeshiva University BRSG No. 9-526-0522 to M.B.P.

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