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()(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; (10) ), is expressed by immature CD4CD8 and CD4CD8, and by mature CD4CD8 and CD4CD8 thymocytes. Two additional voltage-gated K channels, the l-type (Kv3.1, 35) and the n`-type currents (gene unknown), have been found in mature CD4CD8 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 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 channels in CD4CD8 thymocytes. We report the expression of Kv1.1 channels in CD4CD8 thymocytes; these channels have not previously been reported in any lymphoid cell type. Both Kv1.1 and Kv1.3 channels are expressed by CD4CD8 thymocytes, and each of these conductances appears to play a role in early thymocyte development, specifically thymocyte proliferation.

MATERIALS AND METHODS

Cell Isolation

Fetal Thymic Organ Culture

Thymocyte Phenotype Determination

Electrophysiologic Recording

RNA Preparation and Reverse Transcriptase PCR

RESULTS

Pharmacological Characterization of Potassium Currents in CD4CD8Thymocytes-To identify the voltage-dependent K channels expressed by CD4CD8 (double negative, DN) thymocytes, we employed K 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 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 TCR-positive 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) as previously reported(12) . Fig. 1shows a typical family of whole cell currents from an adult CD4CD8 thymocyte evoked by step depolarizations to potentials between -40 and +40 mV (20 mV steps). Potassium currents activated at approximately -40 mV and showed typical time-dependent activation and inactivation characteristics, similar to previous recordings from CD4CD8 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 CD4CD8 thymocytes were comparable, but there was a substantially greater current density in the adult cells. The current density was 262 ± 25.2 µA/cm in adult (n = 29) and 95.4 ± 8.5 µA/cm in fetal cells (n = 30) for a voltage step from -80 to +50 mV.

Figure 1: Voltage-dependent potassium currents in CD4CD8 thymocytes. Top, currents evoked from an immunocytochemically identified CD4CD8 murine thymocyte by 250 ms voltage-clamp steps from -80 mV to from -40 to 40 mV (20 mV steps). The cell was recorded using the nystatin method (Rs = 53 M; Cm = 2.74 pF). Each trace represents the average of currents from two consecutive voltage-clamp steps. Currents activated and inactivated in a time and voltage-dependent manner. Bottom, the peak current/voltage relationship for the total macroscopic currents shown above. Cell number 09089402.

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 -80 to +50 mV before and after toxin addition. Previous reports have indicated that CD4CD8 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 ± 2.7% (n = 6) of the original peak current in adult and 17.9 ± 3.8% (n = 6) in fetal cells. For adult CD4CD8 thymocytes, the peak CTX-resistant current was 60.4 ± 12.9 pA (n = 6), whereas for fetal cells the current was 36.3 ± 5.2 pA (n = 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 current similar to that observed in CD4CD8 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 ± 9.8 pA, n = 7, Fig. 2B); the DTX- resistant current was then blocked by CTX (50 nM, Fig. 2B). Results from a third subtype-specific 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, (13) ), did not block all K current in CD4CD8 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 ± 2.5% for adult and 16.7 ± 2.0% for fetal thymocytes. For adult CD4CD8 thymocytes, the peak KTX-resistant current was 115.4 ± 14.1 pA (n = 5), whereas for fetal cells the current was 40.3 ± 3.8 pA (n = 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).

Figure 2: Pharmacological separation of potassium currents in CD4CD8 thymocytes. The sensitivity of voltage-dependent potassium currents to charybdotoxin and dendrotoxin was determined. K currents were elicited with 250 ms voltage-steps from a holding potential of -80 mV to +50 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 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 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 CTX-resistant K current was 98 pA, and no substantial current remained after subsequent addition of DTX. Cell number 09220401. B, an adult CD4CD8 thymocyte was subjected to voltage pulses from -80 mV holding potential to +50 mV. All parameters were as described for the thymocyte above. The capacitance of this thymocyte was 2.5 pF. The peak control K current was 543 pA. After the addition of DTX the peak current was 472 pA. In this experiment a small residual K current was observed after addition of 100 nM CTX. Cell number 08319404.

Figure 3: DTX and KTX block the entire whole cell K current of CD4CD8 thymocytes. A day 15 fetal CD4CD8 thymocyte was subjected to voltage pulses from -80 mV holding potential to +50 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 current was 367 pA. A combination of 100 mM DTX and 10 nM KTX completely blocked the whole cell K current.

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.

Figure 4: The DTX sensitive component of the whole cell K current does not exhibit use-dependent inactivation. An adult CD4CD8 thymocyte was subjected to a single control voltage pulse (leftpanels) or a train of voltage-step pulses (200 ms, 50-ms interval) from -80 mV to +50 mV to induce use-dependent inactivation of the K+ current (middle and righttraces). Control currents were evoked in the absence (topleft) and presence (bottomleft) of 100 nM DTX. The peak K current in the absence of blocker was 293 pA and in the presence of blocker was 260 pA. In the absence of DTX, a small use-independent K current (peak = 64 pA) was observed (topright). 100 nM DTX blocked most of this current (bottomright). The capacitance of this thymocyte was 2.06 pF.

Identification of mRNA for KV1.1 and KV1.3 in Day 15 Fetal Thymocytes

Figure 5: rtPCR confirms that Kv1.3 and Kv1.1 are expressed by day 15 fetal thymocytes. Total cellular RNA was isolated from day 15 fetal thymocytes, which comprise greater than 95% CD4CD8 thymocytes. The RNA was treated with DNase I to remove contaminating genomic DNA (see ``Materials and Methods''). A portion of the RNA was used to generate cDNA (+ lanes) and subjected to PCR with DNA primers specific to the 3` translated and non-translated regions of Kv1.3 (firstlane) and Kv1.1 (thirdlane). A portion of the day 15 derived total cellular RNA was treated identically as that subjected to cDNA synthesis except for the omission of reverse transcriptase (- lanes) and was subjected to PCR with the same DNA primers for Kv1.3 (secondlane) and Kv1.1 (fourthlane). Primary identification of the PCR-amplified products was made on the basis of their correct predicted sizes (Kv1.3, 550 base pairs; Kv1.1, 450 base pairs). Secondary confirmation was obtained by hybridization with P-labeled unique internal DNA primers (see ``Materials and Methods'').

Potassium Channel Blockers Inhibit Proliferation of Fetal Thymocytes

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 1). 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 (>90% 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 CD4CD8 and CD4CD8 immature thymocytes (TCR-negative), both precursors of CD4CD8 thymocytes(15) , and a decrease in the percentage of CD4CD8 thymocytes (Fig. 6). However, as noted previously (Table 1), 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 CD4CD8 thymocytes (Table 2). 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 CD4CD8 thymocytes is decreased, and that CD4CD8 thymocytes are derived from immature proliferating CD4CD8 and CD4CD8 precursor pools suggest that these drugs inhibit proliferation of thymocyte precursors and/or their progression to the CD4CD8 stage. That the combination of CTX and DTX induces a greater decrease in yield and the number of CD4CD8 thymocytes by day 2 than CTX or DTX alone suggests that they act synergistically.

Figure 6: K channel blockers inhibit fetal thymocyte development. Day 15 fetal thymic lobes were cultured as described in methods in the absence of blocker, or in the presence of 200 nM DTX, 200 nM CTX, or 200 nM DTX plus 200 nM CTX. 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 CD4CD8 and CD4CD8 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 + CTX, respectively. These data are representative of at least six separate experiments with each blocker.

DISCUSSION

We have identified a K conductance not previously described in CD4CD8 thymocytes, which is encoded by the Kv1.1 gene. Patch-clamp studies demonstrated that the predominant channel in CD4CD8 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 CD4CD8 thymocytes is not known.

We have also identified a developmental role for thymocyte potassium channels. Blockers of each of the identified voltage-dependent 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 channel toxins is primarily due to a decrease in CD4CD8 thymocytes. Since at least one proliferative cycle appears to be necessary for maturation of DN thymocytes to the CD4CD8 stage(14) , by blocking cell cycle progression, K channel toxins may prevent maturation of some DN thymocytes to the CD4CD8 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 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 1). 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 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 CD4CD8 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 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 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 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 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.

FOOTNOTES

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by National Institutes of Health Grant HD-01056. To whom correspondence should be addressed.

¶

Supported by National Institutes of Health Grant A132748. Scholar of the Leukemia Society of America.

**

Supported by National Institutes of Health Grant AR-39910.

§§

Supported by National Institutes of Health Grant HL-41084.

()

The abbreviations used are: IL2, interleukin 2; FTOC, fetal thymic organ culture; , ohm(s); F, farad(s); DN, double negative; CTX, charybdotoxin; DTX, dendrotoxin; KTX, kaliotoxin; TCR, T cell receptor; PCR, polymerase chain reaction.

ACKNOWLEDGEMENTS

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