The basic approach

To accomplish all of these goals, we produced stable mammalian cell lines with channel expression under the control of an inducible promoter [1–4]. Any cell line that is good for heterologous expression would be suitable for creating a stable cell line. In addition, the cell line of choice must be amenable to the inducible promoter system being used (c.f. [4]). We used CHO (Chinese hamster ovary) cells due to the extremely low expression level of endogenous ion channels [5]. We purchased T-REx-CHO cells from Invitrogen Corp. (Carlsbad, CA). These cells are stably transformed with the pcDNA6/TR vector, which contains the tetracycline (Tet) repressor gene. We further transformed these cells with a pcDNA4/TO vector, which contained 1) a CMV promoter with two copies of the Tet operator 2 sequence, and 2) the cDNA encoding the channel gene of choice ligated into the multiple cloning site. Channel expression levels were then under the control of a competition between constitutively expressed Tet repressor protein and exogenously added tetracycline. In the absence of Tet, the Tet repressor protein is bound to the Tet operator and expression is prevented. Incubation of cells with Tet results in the displacement of the Tet repressor protein from the Tet operator, and expression occurs.

Production of current densities suitable for macroscopic current recordings

Figure 1 illustrates the time-dependent increase in expression with Tet incubation. Cells were incubated in Tet for the time denoted on the abscissa. Cells were then removed from the incubator and transferred to recording media at room temperature. Whole cell patch clamp recordings were made shortly thereafter to determine current magnitude, and thus, expression level. Fig. 1A illustrates whole cell current magnitude as a function of the duration of Tet incubation. Fig. 1B illustrates the current magnitude normalized for cell size. There was little expression during the first hour, after which there was a steady increase as a function of incubation time. The ideal current magnitude for whole cell voltage clamp recordings was achieved with 2 to 3 hours of incubation (Fig. 1A, inset). (The data in Fig. 1 represent 76 cells recorded over a period of three weeks. On each experimental day, currents were recorded following Tet exposure at several intervals that covered the entire range of Tet exposure duration.)

In the experiment illustrated by Fig. 2A, cells were incubated in Tet for 250 min and then removed from the incubator and placed in recording media. Cells were tested for current expression immediately and every few minutes for ~80 min. These data demonstrate that once cells were removed from the incubator and placed in recording media at room temperature, channel expression levels remained constant (Fig. 2A). This is of some practical significance, as this means that one need not rush to record once channels are removed from the incubator. In contrast, the increase in channel expression continued if Tet exposure was discontinued but cells were maintained in the 37° incubator (Fig. 2B). Thus, whether cells were exposed to Tet for 60 min or 250 min, current density recorded at 250 min was identical (Fig. 2B). This is also convenient, as it demonstrates that channel expression can be tightly controlled without the experimenter having to worry about removing the Tet from the cells at a precise time. If, however, experiments are to take place for more than ~4 hours after the initiation of Tet incubation, the duration of Tet exposure will influence current density at the time of recording. For example, 460 minutes after initiation of Tet incubation, cells had a higher expression level if Tet exposure was maintained rather than terminated after just 60 min (Fig. 2C). Practically speaking, these results suggest that for a full day's worth of experiments, control over expression will be better maintained if Tet incubation is initiated at least twice.

Thirty minutes appeared to be the borderline duration of Tet incubation required for initiation of expression (Fig. 2B). After a 30 minute incubation with Tet, 3/7 cells expressed no current at all above background. In contrast, after exposure to Tet for 60 minutes or more, 100% of cells expressed a current level above background.

Production of current densities suitable for single channel recordings

Figures 1 and 2A,2B,2C demonstrate that current magnitude can be kept within an ideal range for whole cell patch clamp recordings. For recording single ion channel currents, we needed to reliably obtain much lower channel densities. Cells maintained in normal FBS without added Tet displayed small but measurable currents (Fig. 2D). When cells were maintained in Tet-free media (see Methods) for up to 4 weeks, the observed current magnitude was reduced to <112 pA per cell at 0 mV. Normalized for cell size, these currents reflected a current density of less than 10 pA/pF, or <1 channel per μm² (Fig. 2D). This density is well suited to recording currents through single channels with a standard, low resistance patch pipet, which has a tip diameter of 0.8 – 1 μm. (It should be noted that this minimal level of expression is likely to vary with different cell clones.)

A potential problem for single channel recordings is a finite, albeit low level of expression of endogenous channels in the cell line being used. Indeed, we have never observed endogenous, voltage-gated K⁺ currents in CHO cells. Nonetheless, in Fig. 3, we used functional tests to determine whether the current that remained following prolonged incubation in Tet-free medium was predominantly from endogenous channels, the transfected channel, or a mix of the two. We used two well-characterized functional assays on two channels, the wild type Kv2.1 potassium channel and a mutant Kv2.1 channel that had two outer vestibule lysines neutralized (Kv2.1 K356G K382V). These two channels display different sensitivity to the extracellular channel blocker, tetraethylammonium (TEA) [6] and display different responsiveness to elevation of external [K⁺] [7]. When over-expressed in HEK cells, these two channels are blocked by 3 mM external TEA by ~44% and ~81%, respectively ([6]; Fig. 3B). The TEA block of current in CHO cells after prolonged incubation in Tet-free medium was statistically identical for the respective, stably expressed channels (Fig. 3A,3B). Similar results were obtained for the K⁺-dependent change in current magnitude. When expressed in HEK cells, currents through Kv2.1 are potentiated by elevation of external [K⁺] ([7]; Fig. 3D). In contrast, currents through the mutant channel are reduced by the same elevation of external [K⁺] ([7]; Fig. 3D). The currents in CHO cells after prolonged incubation in Tet-free medium responded to [K⁺] elevation in a quantitatively identical manner (Fig. 3C,3D). These data demonstrate that the vast majority, or perhaps all, of the current observed in Tet-free conditions, which was produced by fewer than 100 channels in the entire cell membrane, was carried by the transfected channel.

Single channel recordings

As described above, one of the primary goals of this approach was to achieve expression levels suitable for recording single channel currents in mammalian cells. Fig. 4A and 4B illustrate the problem to be solved. Our standard low resistance patch clamp pipet, which has good electrical properties, and which we would use for whole cell recordings, has a tip diameter of ~0.8 -1 μm. Fig. 4B illustrates a recording with a low resistance pipet from a cell-attached patch on a cell expressing Kv2.1 K356G K382V. This cell was incubated with Tet for 120 min. Shortly after this recording was made, the membrane patch was ruptured, and the whole cell current illustrated in Fig. 4A was recorded. Clearly, even at expression levels that resulted in whole cell currents of just 200 pA (current density ~30 pA/pF), it would be impossible to conduct a comprehensive study of single channel behavior. In contrast, the lower current levels achieved with Tet-free media are ideal for reliably recording single channel currents with low resistance pipets (Fig. 4C). In 51 cells examined thus far, 36 cells displayed either 1 or 2 single channels in a patch.

The data in Fig. 3 demonstrated that few, if any, endogenous voltage-gated K⁺ channels were present in the stable CHO cell lines. Three observations strongly indicated that single channel currents obtained from stably transfected CHO cells also represented exclusively the transfected channels. First, previous whole cell recordings demonstrated that, when normalized for channel number, current magnitude in wild type Kv2.1 was half that of the mutant Kv2.1 channel [8]. The single channel data obtained from the stably transfected CHO cells were consistent with these results. At 0 mV, in the presence of 0 mM external K⁺, single channel current magnitude in CHO cells containing wild type Kv2.1 averaged 0.35 ± 0.02 pA (n = 3). In cells containing Kv2.1 K356G K382V, single channel current magnitude averaged 0.72 ± 0.01 pA (n = 3). Second, under a variety of different experimental conditions, populations of cells transfected with either of the two channels displayed single channel conductances that varied by 5% or less (data not shown). Third, in patches with multiple channels, all single channel events displayed identical conductances. Thus, whereas care must always be taken, it appears that this system allows for the reliable recording of single channel currents from transfected cells with a low probability of contamination from endogenous channels.

Selection and maintenance of the stable cell line and control of expression

Stable cell lines were made for two voltage-gated K⁺ channels, wild type Kv2.1 and a mutant channel derived from Kv2.1 (Kv2.1 K356G K382V). For each channel type, 10 of ~40 cell foci that remained following transfection and exposure to antibiotics were selected and expanded. Four of the ten cell cultures for each channel were then tested for Tet-inducible channel expression. Seven of these eight cultures displayed Tet-induction, and displayed similar macroscopic current magnitudes in both the absence and presence of tetracycline.

Stable cell lines were kept under selective pressure by blasticidin (pcDNA6 contained the blasticidin resistance gene) and Zeocin (pcDNA4 contained the Zeocin resistance gene). Cells were maintained in a CO₂ incubator (37°C, 8.5% CO₂) in Ham's F12 media containing 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 1% penicillin-streptomycin, 10 μg/ml blasticidin and 250 μg/ml Zeocin. In some experiments, cells were maintained in Tet-free FBS (Hyclone Laboratories). Tet-free FBS contained < 300 pg/ml of Tet, determined by ELISA assays (Hyclone Laboratories). Normal FBS was unscreened for Tet. To initiate expression, Tet was added to the maintenance media at a concentration of 1 μg/ml. Expression levels were controlled by the duration of Tet incubation, as described in the Results section.

Electrophysiology

Channel expression was determined using standard whole cell patch clamp recordings, as described previously [9]. Briefly, cells were removed from the incubator, and incubation media was replaced with a bath solution containing, in mM: 145 N-methylglucamine (NMG), 2 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose, pH adjusted with HCl to 7.3, osmolality 330. The intracellular solution contained, in mM: 100 KCl, 35 NMG, 10 EGTA, 4 MgCl₂, 1 CaCl₂, 10 HEPES, pH adjusted with HCl to 7.3, osmolality 290. Once seal formation was achieved, cells were superfused by constantly flowing bath solution. Cells were clamped at a holding potential of -80 mV and currents activated by depolarization to 0 mV. In Fig. 4B and 4C, currents were recorded in the cell-attached patch configuration. In these experiments, the pipet contained intracellular solution, except that 100 mM NMG-Cl was substituted for KCl. The bath solution had 100 mM KCl substituted for NMG-Cl. This held the whole cell resting potential near 0 mV (data not shown), which enabled us to know the membrane potential across the patch. In cell attached patches, the pipet potential with respect to ground was held at +80 mV, and channels were activated by stepping the pipet potential to 0 mV. All currents were filtered at 2 KHz (Axopatch 200B amplifier, Axon Instruments, Union City, CA) and digitized at 10 KHz for whole cell recordings and 20 KHz for single channel recordings (pClamp 9, Axon Instruments). Recording pipets were fabricated from 8250 glass (Garner Glass, Claremont, CA), coated with Sylgard and firepolished. Pipet resistance was between 1.5 and 2.0 MΩ.