Identification of Kv1.1 expression by murine CD4-CD8- thymocytes. A role for voltage-dependent K+ channels in murine thymocyte development.

The patch-clamp recording technique and RNApolymerase chain reaction were used to identify the voltage-dependent K+ channels expressed by murine fetal and adult CD4−CD8− thymocytes. Two distinct currents, encoded by the genes Kv1.1 and Kv1.3 were identified based upon their biophysical and pharmacologic characteristics and confirmed with RNA-polymerase chain reaction. Peptide blockers of Kv1.1 and Kv1.3 gene products were also applied to a murine fetal thymic organ culture system to investigate the developmental role of these K+ channels. Dendrotoxin (DTX) and charybdotoxin (CTX), antagonists of Kv1.1 and Kv1.3 channels, respectively, decreased thymocyte yields in organ culture without affecting thymocyte viability. DTX-treated thymi contained 56 ± 8% (n = 8 experiments), and CTX-treated thymi contained 74 ± 4% (n = 7 experiments) as many thymocytes as untreated lobes. DTX and CTX also altered the developmental progression of thymocytes in fetal organ culture. These data provide the first evidence of Kv1.1 expression in a lymphoid cell and indicate that thymocyte voltage-dependent K+ channels are critical to thymocyte preclonal expansion and/or maturation.

Membrane potassium channels subserve important physiologic functions of thymocytes and peripheral T lymphocytes. Although lymphocytes are not electrically excitable cells, they express potassium channels that are the primary determinants of the membrane potential (1,2), and are critically important for production of the requisite T cell growth factor IL2 1 (3)(4)(5). Three voltage-dependent potassium channels have been described in murine thymic lymphocytes (6,7). These currents were originally distinguished on the basis of their unique pharmacology and kinetic behavior (6). The most prevalent, n-type (Kv1.3 gene product; Ref. 10), is expressed by immature CD4 Ϫ CD8 Ϫ and CD4 ϩ CD8 ϩ , and by mature CD4 ϩ CD8 Ϫ and CD4 Ϫ CD8 ϩ thymocytes. Two additional voltage-gated K ϩ channels, the l-type (Kv3. 1,35) and the nЈ-type currents (gene unknown), have been found in mature CD4 Ϫ CD8 ϩ thymocytes only (6).
Although the physiologic role of voltage-gated potassium channels during normal thymocyte development (and preclonal expansion) has not been characterized, the selective expression of specific K ϩ channel subtypes by thymocyte subpopulations suggests that these channels may play a critical role in thymocyte development. We have used the fetal thymic organ culture (FTOC) system to study thymic development and have employed molecular biological and patch-clamp recording techniques to determine the expression of thymocyte K ϩ channels in CD4 Ϫ CD8 Ϫ thymocytes. We report the expression of Kv1.1 channels in CD4 Ϫ CD8 Ϫ thymocytes; these channels have not previously been reported in any lymphoid cell type. Both Kv1.1 and Kv1.3 channels are expressed by CD4 Ϫ CD8 Ϫ thymocytes, and each of these conductances appears to play a role in early thymocyte development, specifically thymocyte proliferation.

MATERIALS AND METHODS
Cell Isolation-Virus-free C57Bl/6 mice were obtained from Jackson Laboratories or the National Cancer Institute and were bred in the animal facility at the University of Pennsylvania. Thymic lobes were removed from fetal mice at desired gestational ages and were placed into RPMI medium containing 10% heat-inactivated fetal bovine serum, 50 mM 2-mercaptoethanol, 10 mM HEPES buffer, pH 7.4, 1 mM L-glutamine, and 100 IU/ml penicillin and streptomycin. Thymocytes were isolated by teasing apart the lobes with sterile keratotomy knives.
Fetal Thymic Organ Culture-FTOC (8) was established in 24-well plates at 37°C in a 5%CO 2 /95%O 2 environment. Surgical gel foam (1-cm 2 ; Upjohn) was soaked in complete RPMI medium to which K ϩ blockers or additional reagents were added. Fetal lobes were placed onto sterile circular nitrocellulose filters (Millipore), which were supported by the gel foam. The fetal lobes (3/filter) were maintained at the air/medium interface and were bathed with medium daily. One half of the culture medium was changed on alternate days.
Electrophysiologic Recording-Murine fetal or adult DN thymocytes stained with fluorescein (CD8)-or phycoerythrin (CD4)-tagged monoclonal antibodies (see above) were placed in a recording chamber (Brooks) mounted on an inverted fluorescent microscope (Diaphot, Nikon) and CD4 Ϫ CD8 Ϫ (non-staining) cells were patched. Patch pipettes (4 -6 M⍀) were back-filled with solution containing 150 g/ml nystatin * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  (Sigma). Command potentials were generated and currents acquired, stored, and analyzed on a Macintosh computer using an EPC-9 amplifier and associated software (Heka, Lambrecht, Germany). Liquid junction potentials, capacitance, and series resistance were compensated using the internal circuit of the EPC-9. Data were sampled at 10 kHz, filtered at 1 kHz, and recorded on computer disk. Following formation of gigaohm seals (5-30 G⍀), cells were lifted off the chamber bottom and held at Ϫ80 mV; voltage clamp measurements were initiated when the access resistance (R s ) was less than 50 M⍀ (ϳ5-10 min after seal formation). Unless stated otherwise, macroscopic K ϩ currents were elicited with step depolarizations to ϩ50 mV and currents reported as peak whole cell currents. Current density was calculated by dividing the peak whole cell current by the cell capacitance, with the assumption that 1 m 2 ϭ 1 pF. Drugs were applied by direct addition to the bath. The bath solution contained (in mM) 160 NaCl, 4.5 KCl, 2 MgCl 2 , 1 CaCl 2 , 5 Na-HEPES, and 5 glucose (pH 7.4). The pipette solution contained 134 KF, 11 K 2 EGTA, 1.1 CaCl 2 , 2 MgCl 2 , and 10 K-Hepes (pH 7.2). Synthetic charybdotoxin (CTX) and kaliotoxin (KTX) were obtained from Peptides International (Louisville, KY), and dendrotoxin (DTX) from Calbiochem (San Diego, CA).
RNA Preparation and Reverse Transcriptase PCR-Total cellular RNA was prepared according to the method of Chomczinski and Saachi (9). Contaminating genomic DNA was removed from RNA preparations by treating them with DNase I (Boerhinger Mannheim) before cDNA synthesis. The polymerase chain reaction was used to amplify cDNA prepared from murine thymocyte RNA. Unique oligonucleotide primer pairs were designed to 3Ј regions of the K ϩ channel genes as follows: . PCR products were separated by electrophoresis in a 2% agarose gel (Life Technologies, Inc.) and capillary blotted onto GeneScreen Plus nylon membrane (DuPont NEN) with 0.5 M NaOH and 1.5 M NaCl. Oligonucleotide probes were 5Ј-end-labeled with [␥-32 P]ATP using T4 polynucleotide kinase (Life Technologies, Inc.).

Pharmacological Characterization of Potassium Currents in CD4
Ϫ CD8 Ϫ Thymocytes-To identify the voltage-dependent K ϩ channels expressed by CD4 Ϫ CD8 Ϫ (double negative, DN) thymocytes, we employed K ϩ channel-selective peptide blockers with the nystatin variation of the whole cell patch-clamp technique. Immunocytochemically defined fetal (day 15 or 16) or adult murine thymocytes were voltage-clamped and K ϩ currents evoked by step depolarizations. Experiments were performed on both fetal DN thymocytes, which are predominately TCR-negative, and adult DN cells, which are both ␣␤TCRpositive and -negative (16). Current amplitudes were stable in these cells for more than 45 min; the rate of current inactivation did accelerate somewhat over time, however, and was dependent on the series resistance (R s ) as previously reported (12). Fig. 1 shows a typical family of whole cell currents from an adult CD4 Ϫ CD8 Ϫ thymocyte evoked by step depolarizations to potentials between Ϫ40 and ϩ40 mV (20 mV steps). Potassium currents activated at approximately Ϫ40 mV and showed typical time-dependent activation and inactivation characteristics, similar to previous recordings from CD4 Ϫ CD8 Ϫ cells (6, 7). Current inactivation was incomplete, however, and a small non-inactivating current component was a consistent feature of the currents in these cells. Total currents found in fetal and adult murine CD4 Ϫ CD8 Ϫ thymocytes were comparable, but there was a substantially greater current density in the adult cells. The current density was 262 Ϯ 25.2 A/cm 2 in adult (n ϭ 29) and 95.4 Ϯ 8.5 A/cm 2 in fetal cells (n ϭ 30) for a voltage step from Ϫ80 to ϩ50 mV.
Whole cell currents were pharmacologically characterized with several potassium channel-selective peptidyl toxins; outward currents were evoked by 250 or 500 ms depolarizing voltage steps from Ϫ80 to ϩ50 mV before and after toxin addition. Previous reports have indicated that CD4 Ϫ CD8 Ϫ thymocytes express Kv1.3 channels, which are completely blocked by CTX (6), which blocks Kv1.2, Kv1.3 and Kv1.6 gene products (11,13,36). We consistently observed a small, CTX-resistant (100 nM) current in fetal and adult cells, as shown in Fig. 2A.
The CTX-insensitive current was 8.4 Ϯ 2.7% (n ϭ 6) of the original peak current in adult and 17.9 Ϯ 3.8% (n ϭ 6) in fetal cells. For adult CD4 Ϫ CD8 Ϫ thymocytes, the peak CTX-resistant current was 60.4 Ϯ 12.9 pA (n ϭ 6), whereas for fetal cells the current was 36.3 Ϯ 5.2 pA (n ϭ 6). The CTX-resistant current was blocked by DTX (100 nM), an inhibitor of Kv1.1, Kv1.2, and Kv1.6 channels (11). The only gene known to encode a CTX-insensitive, DTX-sensitive, voltage-gated K ϩ current similar to that observed in CD4 Ϫ CD8 Ϫ thymocytes is Kv1.1. The CTX-resistant current is also completely blocked by 0.8 mM tetraethylammonium (data not shown). Consistent with the presence of a charybdotoxin-insensitive current other than Kv1.3, the initial application of DTX to double negative thymocytes blocked a small component of the peak current (74 Ϯ 9.8 pA, n ϭ 7, Fig. 2B); the DTX-resistant current was then blocked by CTX (50 nM, Fig. 2B). Results from a third subtypespecific toxin were also consistent with the presence of a Kv1.1 channel current. KTX, which is selective for Kv1.3 at the concentration used (10 nM, Ref. 13), did not block all K ϩ current in CD4 Ϫ CD8 Ϫ thymocytes. As with experiments using CTX and DTX, a small KTX-resistant current was observed (data not shown). Moreover, similar to experiments with CTX, KTX completely blocked outward current in the presence of DTX (Fig. 3).
The KTX-resistant current was 18.5 Ϯ 2.5% for adult and 16.7 Top, currents evoked from an immunocytochemically identified CD4 Ϫ CD8 Ϫ murine thymocyte by 250 ms voltage-clamp steps from Ϫ80 mV to from Ϫ40 to 40 mV (20 mV steps). The cell was recorded using the nystatin method (Rs ϭ 53 M⍀; Cm ϭ 2.74 pF). Each trace represents the average of currents from two consecutive voltageclamp steps. Currents activated and inactivated in a time and voltagedependent manner. Bottom, the peak current/voltage relationship for the total macroscopic currents shown above. Cell number 09089402. Ϯ 2.0% for fetal thymocytes. For adult CD4 Ϫ CD8 Ϫ thymocytes, the peak KTX-resistant current was 115.4 Ϯ 14.1 pA (n ϭ 5), whereas for fetal cells the current was 40.3 Ϯ 3.8 pA (n ϭ 9). Thus subtype-specific toxins demonstrate a DTX-sensitive and CTX/KTX-resistant current, consistent with the expression of Kv1.1 channels, as suggested by the expression of Kv1.1 mRNA (see below).
In addition to their unique pharmacologies, Kv1.1 and Kv1.3 are biophysically distinct in that Kv1.3 exhibits use-dependent inactivation, whereas Kv1.1 does not (13). As shown in Fig. 4, repeated depolarizations at short intervals (4 Hz) resulted in the progressive inactivation of one current component, which was DTX-insensitive. Conversely, a non-cumulatively inactivating current component existed, which was blocked by DTX. Similar to results with application of CTX, KTX, or DTX, the inactivating current (presumably Kv1.3) was the predominant current component.

Identification of mRNA for KV1.1 and KV1.3 in Day 15
Fetal Thymocytes-RNA-PCR was used to confirm the presence of mRNA in CD4 Ϫ CD8 Ϫ thymocytes for K ϩ channels. Fetal thymocytes from day 15 gestation were used because they are almost exclusively CD4 Ϫ CD8 Ϫ . Using channel subtype-specific primers for Kv1.1 through Kv1.6, and for all members of the Kv2, Kv3, and Kv4 families, only PCR products of the appropriate size for Kv1.1 and Kv1.3 were obtained. Fig. 5 is an autoradiograph of a Southern blot of PCR products generated from Kv1.3 (lane 1) and Kv1.1 (lane 3) mRNA. The identity of the amplified cDNA was confirmed by hybridization with radiolabeled internal oligonucleotides specific for each of these genes (see "Materials and Methods"). Because these K ϩ channel genes are intronless (10), we confirmed that these PCR products were not amplified from contaminating K ϩ channel genomic DNA in the RNA samples. These data indicate that no PCR products were obtained for Kv1. 3 FIG. 3. DTX and KTX block the entire whole cell K ؉ current of CD4 ؊ CD8 ؊ thymocytes. A day 15 fetal CD4 Ϫ CD8 Ϫ thymocyte was subjected to voltage pulses from Ϫ80 mV holding potential to ϩ50 mV. All parameters were as described for the thymocyte in Fig. 2. The capacitance of this thymocyte was 2.8 pF. The peak control K ϩ current was 367 pA. A combination of 100 mM DTX and 10 nM KTX completely blocked the whole cell K ϩ current.

FIG. 2. Pharmacological separation of potassium currents in
CD4 ؊ CD8 ؊ thymocytes. The sensitivity of voltage-dependent potassium currents to charybdotoxin and dendrotoxin was determined. K ϩ currents were elicited with 250 ms voltage-steps from a holding potential of Ϫ80 mV to ϩ50 mV. A, each curve represents the average of two consecutive stimuli 30 s apart. Three superimposed responses represent stable currents obtained from the same cell (from largest to smallest current) in the absence of K ϩ blocker, in the presence of 50 nM CTX, and in the presence of 50 CTX and 100 DTX. These data are representative of six separate experiments, which demonstrate CTX-sensitive, and CTX-resistant and DTX-sensitive components of the whole cell K ϩ current. The experiment was conducted at 20 -24°C under standard conditions (see "Materials and Methods"). The capacitance of this cell was 1.8 pF. The peak control current was 694 pA. The peak CTXresistant K ϩ current was 98 pA, and no substantial current remained after subsequent addition of DTX. Cell number 09220401. B, an adult CD4 Ϫ CD8 Ϫ thymocyte was subjected to voltage pulses from Ϫ80 mV holding potential to ϩ50 mV. All parameters were as described for the thymocyte above. The capacitance of this thymocyte was 2.5 pF. The peak control K ϩ current was 543 pA. After the addition of DTX the peak current was 472 pA. In this experiment a small residual K ϩ current was observed after addition of 100 nM CTX. Cell number 08319404.

Potassium Channel Blockers Inhibit Proliferation of Fetal
Thymocytes-Cellular expansion is a dominant feature of thymocyte development between days 15 and 17 of gestation, and cell cycle activity may be requisite for subsequent development to the CD4 ϩ CD8 ϩ phenotype (14). To determine whether Kv1.1 and/or Kv1.3 play a critical role during development of thymocytes in situ, we characterized the effects of K ϩ channel blockers on thymocyte development in whole cultured day 15 fetal thymic explants. In situ blockade of Kv1.3 was achieved with CTX or KTX and of Kv1.1 with DTX. To minimize variability within individual experiments, at least two identically treated thymic lobes were pooled and used for yield determinations and phenotype analysis.
The most dramatic effect of the blockers was on total thymocyte yields. The cell yields from treated thymi were lower than in control thymi; however, the inhibitory effect of CTX on yield was consistently less than that of the other blockers (Table I). Because cultured thymic explants do not receive additional thymocyte progenitors, an observed increase in the number of thymocytes within a lobe during culture must necessarily result from proliferative expansion of those thymocytes already in the thymus. Conversely, any decrease is indicative of either inhibition of proliferation or toxicity. Apparent thymocyte viability was unaffected by any of the blockers (Ͼ90% for all treatments). Moreover, neither CTX or DTX were directly toxic and the viability and CD4/CD8 phenotype of murine thymocytes incubated in suspension culture for 48 h with CTX (200 nM) or DTX (200 nM) was not different from control cultures (data not shown). Hence, the reduction in cell yield is probably due to inhibition of proliferation.
In addition to the effects of CTX and DTX on thymocyte yields, we observed an effect on the CD4/CD8 phenotype of thymocytes in FTOC. Treatment of FTOC with CTX, DTX, or both together caused an increase in the percentage of CD4 Ϫ CD8 Ϫ and CD4 Ϫ CD8 ϩ immature thymocytes (TCR-negative), both precursors of CD4 ϩ CD8 ϩ thymocytes (15), and a decrease in the percentage of CD4 ϩ CD8 ϩ thymocytes (Fig. 6).
However, as noted previously (Table I), we consistently observed CTX to have less effect than DTX on thymocyte yield. The reduction in cell yield induced by DTX or DTX and CTX is primarily a reflection of a reduction in the absolute number of CD4 ϩ CD8 ϩ thymocytes (Table II). Even though the effect of CTX alone on yield is less than DTX, its effect on phenotype is similar. Taken together, our observations that CTX and DTX are not directly toxic to thymocytes, that the number of CD4 ϩ CD8 ϩ thymocytes is decreased, and that CD4 ϩ CD8 ϩ thymocytes are derived from immature proliferating CD4 Ϫ CD8 Ϫ and CD4 Ϫ CD8 ϩ precursor pools suggest that these drugs inhibit proliferation of thymocyte precursors and/or their progression to the CD4 ϩ CD8 ϩ stage. That the combination of CTX and DTX induces a greater decrease in yield and the number of Thymocytes isolated on day 2 of culture were stained for surface CD4 and CD8 and analyzed on a flow cytometer (Becton Dickinson). Plots are displayed with CD4 fluorescence on the ordinate and CD8 fluorescence on the abscissa, and the individual thymocyte subpopulations are enclosed within rectangular gates. The percentage of thymocytes within each gate is indicated in the outside corner. This is one of three similar experiments showing that the percentage of CD4 Ϫ CD8 Ϫ and CD4 Ϫ CD8 ϩ thymocytes is increased by each of the blockers. However, each blocker also decreased thymocyte yields compared with untreated controls. For this experiment, the total number of thymocytes recovered per lobe was 384,300 for untreated thymi and 253,000, 325,905, and 225,600 for thymi treated with DTX, CTX, and DTX ϩ CTX, respectively. These data are representative of at least six separate experiments with each blocker.  CD4 ϩ CD8 ϩ thymocytes by day 2 than CTX or DTX alone suggests that they act synergistically. DISCUSSION We have identified a K ϩ conductance not previously described in CD4 Ϫ CD8 Ϫ thymocytes, which is encoded by the Kv1.1 gene. Patch-clamp studies demonstrated that the predominant channel in CD4 Ϫ CD8 Ϫ thymocytes is Kv1.3, in agreement with previous findings in which the n current predominated (6,7). However, our studies also indicate that Kv1.1 channels make up an appreciable current component. Thus, a DTX-sensitive current component was approximately 10% of the peak current. Unlike the Kv1.3 channel current, the Kv1.1 channel current activated slowly and displayed little time-or use-dependent inactivation. The extent to which the distinct kinetic and inactivation properties of these channels determine the membrane potential and calcium signaling in CD4 Ϫ CD8 Ϫ thymocytes is not known.
We have also identified a developmental role for thymocyte potassium channels. Blockers of each of the identified voltagedependent conductances inhibit proliferation of thymocytes within the thymus, suggesting that voltage-dependent potassium channels contribute to the signals that control early developmental events of thymus cellularity. The decrease in the absolute number of thymocytes by K ϩ channel toxins is primarily due to a decrease in CD4 ϩ CD8 ϩ thymocytes. Since at least one proliferative cycle appears to be necessary for maturation of DN thymocytes to the CD4 ϩ CD8 ϩ stage (14), by blocking cell cycle progression, K ϩ channel toxins may prevent maturation of some DN thymocytes to the CD4 ϩ CD8 ϩ stage. The characteristics of DTX and CTX mediated effects on the thymus are different, suggesting that they interact with different targets. The action of DTX is probably mediated by Kv1.1, although at the concentration used in FTOC experiments, DTX would also block Kv1.3 by approximately 40%. Since stromal cells are critical to thymocyte development (34), we cannot rule out the possibility that the effects of these blockers on thymocyte development are mediated by stromal cells within the thymus. However, the effects of DTX and CTX on thymocyte development that we observe are consistent with their interaction with K ϩ channels expressed by thymocytes. It should be noted that, whereas the electrophysiologic experiments indicate that CTX-sensitive Kv1.3 channels constitute the major current at strongly depolarized potentials, CTX, KTX, and DTX had similar effects on thymocyte yield (Table I). This may indicate that the available current at strongly depolarized potentials may not accurately reflect activity at physiological membrane potentials, or that regulation of a particular K ϩ channel in vivo is more relevant to its physiological function.
Several physiological roles have been defined for potassium channels in lymphoid cells. Voltage-dependent potassium channels are critical determinants of the transmembrane electrical potential for both thymocytes and peripheral blood T lymphocytes (17)(18)(19)(20). For peripheral T lymphocytes, production of the T cell requisite growth factor (IL2) and the proliferative response to stimulation critically depend upon the transmembrane potential defined by these potassium channels (4,5,21). It is unlikely, however, that the inhibitory effect of potassium blockers on proliferation of CD4 Ϫ CD8 Ϫ thymocytes is related to an effect upon IL2 production, since the production of IL2 is not critical for thymocyte development (22). However, we have not determined whether any cytokines elaborated in the thymus would decrease the sensitivity of thymocytes to potassium channel blockers in situ. In addition to a role in setting or controlling the membrane potential, the plasma membrane K ϩ permeability is a critical component of the volume regulatory behavior of lymphocytes (1,(23)(24)(25)(26). Although the cell volume of thymocytes changes during development, a physiological role for K ϩ channel-mediated volume regulation has not been demonstrated.
In conclusion, our data extend the observations of Lewis and Cahalan (6) and support the hypothesis that differential expression of potassium channel subtypes may play an important role in development (4). This could occur by several mechanisms. For example, modulation of the lymphocyte K ϩ permeability exerts a substantial effect on membrane potential (1, 2), receptor-mediated transmembrane flux of calcium (5,19,(27)(28)(29)(30)(31)(32), and cytokine production (4,5,21,33). Future efforts should be directed toward understanding why thymocytes have evolved such a complex K ϩ channel phenotype and whether this phenotype is critical to the functional requirements of other thymocyte subpopulations during later developmental events such as T cell repertoire selection.

TABLE II
The effect of K ϩ channel blockers on the absolute number of thymocytes in each subpopulation Thymic lobes were cultured in the absence or presence of CTX, DTX, or both (each at 200 nM), and thymic lobes were dissociated on indicated day of culture. The absolute thymocyte yield was determined for each treatment. The number of thymocytes of each indicated phenotype was determined by multiplying the absolute yield by the percentage of thymocytes that possess indicated phenotype as determined by flow cytometry (see Fig. 6).