In this study we have assessed the suitability of an immunomagnetic positive selection technique (REAlease®) for patch-clamp electrophysiology of CD4+ T cells. We showed that the presence of the immunomagnetic beads on the cell surface does not interfere with the cell membrane capacitance measurements, and thus, normalizing the currents on membrane capacitance can be a reliable method to determine current density (pA/pF) of bead-bound cells as well. Moreover, we showed that the presence of the beads on the cell surface does not interfere with the operation of Kv1.3, the predominant voltage-gated K+ channel of T cells. We found that neither the activation and inactivation kinetics of the whole cell Kv1.3 current nor the voltage-dependence of steady-state activation of the channels was sensitive to the presence of the immunomagnetic beads. We have also demonstrated that the equilibrium block of the Kv1.3 channels by the small-molecule inhibitor TEA+ and the peptide toxin ChTx as well as the kinetics of the ChTx block are similar in bead-bound and control, negatively selected T cells. Based on our results we strongly favor the idea that the immunomagnetic cell separation, particularly the MACS® technique is a suitable method for electrophysiology experiments on immune cells.
We demonstrated that immunomagnetic positive and negative separation techniques result in the sample purity as FACS sorting. It is important to highlight that the purity in case of FACS sorting highly depends on the instrument settings (34). In our setting (“purity mode” of FACS) the expected and obtained purities are similar to the data that we achieved using the immunomagnetic separation techniques with the disadvantage of potentially decreased recovery (35). It is also well-known that FACS may have significant effects on cell viability (14) and even minor changes in the experimental conditions have significant effect on the outcomes. With optimal experimental conditions the viability reached via FACS sorting is similar to that of our immunomagnetic sorting (36), as reflected by our results. We have the experience that in many cases, FACS sorted cells (sorted configuration) are much more stressed possibly due to the high shear stress (12, 13) and high voltage (as shown in case of sorting bacteria samples (37)) during the sorting process. The exclusion of patch-clamp records due to technical inappropriateness in case of membrane capacitance and I-V measurements (as described below) was more frequent for FACS sorted cells, however, it did not reach mathematical significance as examined by Fischer-exact test (data not shown). Beside this, we can subjectively conclude that the sealing efficiency (i.e. obtaining high quality seals between the patch pipette and the membrane in the order of GΩ) was also affected in case of sorted configuration, however, there were no statistical analyses made.
The biophysical parameters of Kv1.3 were mostly oblivious to the presence of beads or any REAlease® components, and even if there was a statistically significant difference between τact values describing the kinetics of Kv1.3 current activation, we do not attribute biological significance to this for three reasons. First, the activation kinetics are fast in any of the configurations, more than two orders of magnitude faster than the inactivation kinetics of the currents, i.e., the channels do open well-before inactivation takes place (Fig. 4C), and thus, a negligible change in the activation kinetics should not affect the mean open time of the channels. Second, the τact values were in the normal physiological range characteristic for Kv1.3 (38) in all samples. Third, the activation kinetics of the current in the control sample did not differ from any of the configurations. It is also important to note, that neither the bead-bound nor the bead free configuration was different from the control and sorted configuration in this parameter. This indicates that none of the components of the REAlease® complex modified the activation kinetics of the Kv1.3 current.
Beside these, neither the inactivation kinetics of Kv1.3 current, nor the cell capacitance values of the measured T-lymphocytes showed statistically significant differences however, as we highlighted the FACS-sorted cells showed more discrete cell capacitance (and consequently size) as compared to the MACS-sorted samples (either REAlease® or negative selection). This may be due to the fundamental differences between the two separation techniques; in case of FACS sorting, the routinely applied forward scatter (FSC) gating discriminates the size of cells, whereas in case of immunomagnetic separation techniques, this factor is absent. Our data show that the presence of the magnetic beads does not alter the physical properties of the membrane and its surrounding liquid to be reflected in the membrane capacitance of the cells. Thus, current density, i.e. current normalized to the cell membrane capacitance (pA/pF), commonly used to quantify the expression of ion channels in a given cell, is a valid variable when magnetic bead-based separation is applied.
The V1/2 values – descriptive of the conductance-membrane potential relationship − are consistent with the literature, being in the range described for Kv1.3 in lymphocytes (38, 39). The voltage sensor of voltage-gated ion channels traverses the transmembrane electric field during activation and as such, the gating apparatus is sensitive to multiple factors, among others, the surrounding lipids (40), externally applied charged fatty acids (41), and cell surface charge screening by divalent cations (42). Some of these effects are based on electrostatic interactions with the voltage sensor. Our results indicate, that the presence of paramagnetic beads on the cell surface does not interfere with the voltage sensor apparatus of Kv1.3, i.e. the alterations in the local electrostatic caused by the presence of the beads is not large enough to be resolved using our experimental approach. In conclusion, the voltage dependent gating of Kv1.3 is oblivious to the presence of the beads and thus, the magnetic bead-based separation method is applicable to electrophysiological studies of lymphocytes.
According to our pharmacological measurements, neither the equilibrium block (IC50) of both TEA+ and ChTx nor the off-rate (koff=1/ τoff) of the ChTx is affected by the presence of the beads on cell surface. The lack of effect on IC50 and τoff in case of ChTx indicates, that the wash-in kinetics of the peptide antagonist is also unaffected by the bead-antibody complex presence on the cell surface (43). In summary, our pharmacological experiments clearly showed that the presence of bead-antibody complex on cell surface did not alter either block equilibrium for TEA+ and ChTx or the block kinetics for the large peptide blocker, ChTx. This insensitivity of the blockers to the presence of the beads is different form the used of ~ 30 nm diameter gold particles conjugated to monoclonal antibodies targeting MHCI and MHC II molecules and the α subunit of the IL-2 receptor, where a slower association kinetics of a scorpion toxin (Pi2) and Kv1.3 was observed (22). The insensitivity of Kv1.3 block to the presence of the beads might be important in physiological assays, where Kv1.3 blockers are widely used to inhibit the proliferation of various T cell subsets (44, 45).