The human isoform 3 Kv7.2 (Y15065) and Kv7.3 (NM004519) cDNAs were provided by T. Jentsch (Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany) and the cDNA encoding rat CaM was provided by the group of J.P. Adelman (Vollum Institute, Portland, OR, USA). The subunits tagged at the N-terminal with mCFP or mYFP were cloned into pCDNA3.1 and we previously confirmed that these N-terminal tags have no impact on the electrophysiological properties of the channel (Gómez-Posada et al., 2011). Point mutations were constructed by PCR-based mutagenesis. The Kv7.3 A315T construct has been described previously (Gómez-Posada et al., 2010). The CaM sponge, that has CFP and YFP flanking the apoCaM-binding site of neuromodulin, was provided by D.J. Black (University of Missouri, Missouri, USA), PI(4)P5-kinase type IV was provided by B. Hille (University of Washington, Seattle, USA) and Dr-VSP-IRES-GFP from zebrafish was provided by Y. Okamura (Osaka University, Osaka, Japan).

HEK293T cells (HEK 293T/17, ATCC, CRL-11268) were maintained in 5% CO₂ at 37°C in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich), supplemented with non-essential amino acids (Sigma, Madrid, Spain) and 10% FBS (Lonza, Madrid, Spain).

Whole-cell patch-clamp recordings of HEK293T cells were obtained at RT (21–25°C) 48 h after transfection using lipofectamine 2000 (Invitrogen). Cells were bathed in the following solution (mM): 140 NaCl, 4 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES (Na), 5 D-glucose, adjusted to pH 7.4 with NaOH. The osmolarity was adjusted with mannitol to ~315 mOsm. Pipettes were pulled from borosilicate glass capillaries (Sutter Instruments, USA) using a Narishige micro-pipette puller (PC-10; Narishige Instrument Co., Japan). Membrane currents were measured using an EPC-8 amplifier (HEKA Instruments, Germany) with >80% series resistance compensation, pipette and membrane capacitance cancellation. Cells with similar fluorescent intensity were selected. All experiments to test the impact of DrVSP (0.5 μg cDNA per 35 mm dish) were carried out with 100% series resistance compensation using a VE-2 amplifier (Alembic Instruments, Canada) equipped with Rs Compensator (Sherman et al., 1999).

Pipettes were filled with an internal solution containing (mM): 125 KCl, 10 HEPES (K), 5 MgCl₂, 5 EGTA, 5 Na₂ATP, adjusted to pH 7.2 with KOH and the osmolarity adjusted to ~300 mOsm with mannitol. The amplitude of the Kv7 current was defined as the peak difference in current relaxation measured at −30 mV after 500–1,500 ms pulses to −110 mV (all channels closed) and to +30 mV (all channels opened). This protocol subtracts all leak currents from the measurements. The data were acquired and analyzed using pCLAMP software (version 8.2), normalized in Excel (Microsoft Corp., Madrid, Spain) and plotted using SigmaPlot (SPSS Corp., Madrid, Spain).

Data are shown as the mean ± S.E.M. The differences between the means were evaluated using the unpaired Student's t-test, where values of P ≤ 0.05 were considered significant. The number of cells in each experiment is indicated in brackets in the figures. The results are from two or more independent batches of cells. In all figures ^*, ^**, and ^*** indicate significance at the p < 0.05, p < 0.01, and p < 0.001, respectively.

The impact of CaM elevation on the function of Kv7 channels has been studied before, often with contrasting results (Gamper et al., 2005; Xu et al., 2007; Zaika et al., 2007; Alaimo et al., 2013; Kang et al., 2014). In the present experiments, we observed that CaM co-expression potentiated the maximal current density of Kv7.2 isoform 3 channels expressed in HEK293T cells (Figure 1) and in CHO cells (data not shown) (Ambrosino et al., 2015); similar results were also achieved when the human isoform 4 of Kv7.2 was expressed (data not shown). In these experiments, to monitor the expression of the channel, isoform 3 was tagged at the N-terminus with CFP; this manipulation has been previously shown not to influence channel function, as the current size and the gating properties of tagged subunits were undistinguishable from those of untagged subunits (Soldovieri et al., 2006). We evaluated next the electrophysiological consequences of transfecting increasing amounts of Kv7.2 cDNA in HEK293T cells by whole-cell patch-clamp. The experiments revealed that the Kv7.2 current density was relatively constant irrespective of the amount of Kv7.2 cDNA transfected (Figure 1A), reaching a maximal value of about 75 pA/pF. The density roughly doubled when CaM was co-expressed, attaining a maximum of approximately 150 pA/pF (Figure 1A). The response to increasing amounts of CaM revealed that half of the maximal density was obtained at a 1:2 Kv7.2/CaM cDNA ratio, whereas no significant effect was detected at a 1:1 ratio (Figure 1B; Alaimo et al., 2013).

To address the requirement of Ca²⁺ binding to CaM, the effect of CaM₁₂₃₄ was assessed. CaM₁₂₃₄ harbors D>A substitutions at each of the four EF-hands, preventing Ca²⁺ binding (Putkey et al., 1989). The extents of current potentiation observed with CaM and CaM₁₂₃₄ were indistinguishable (Figures 2A,B), although CaM₁₂₃₄ overexpression caused a leftward shift in the voltage dependence of activation of Kv7.2 channels (Figure 2C). Thus, the increase in current density does not require Ca²⁺ binding to CaM (Ambrosino et al., 2015).

To address the consequences of reduced CaM levels we over-expressed a CaM binding protein (CaM sponge, Figure 3; Black et al., 2004; Liu et al., 2010). This intervention led to a decrease in Kv7.2 current density (Figure 3) and to a right shift in the voltage-dependence (Figure 3C). When both the CaM sponge and CaM₁₂₃₄ were co-expressed the current density was similar to control (data not shown), supporting the notion that the effect of the CaM-sponge (neurogranin) on Kv7.2 was due to reduced CaM availability.

To evaluate the role of PI(4,5)P₂ in Ca²⁺-independent CaM regulation of Kv7.2 currents, we used Danio rerio voltage-dependent phosphatase (DrVSP), which catalyzes the removal of a phosphate group at position 5, resulting in the rapid depletion of PI(4,5)P₂. DrVSP is barely active at the voltages used to quantify Kv7 voltage-evoked current relaxations (Falkenburger et al., 2010), but becomes activated at highly-depolarized voltages (Figure 4A). The consequence of elevation (CaM₁₂₃₄ co-expression) or reduction (CaM sponge co-expression) of CaM levels on DrVSP sensitivity of Kv7.2 currents was investigated (Figure 4). The results obtained revealed that activation of DrVSP was less effective at reducing Kv7.2 currents under both experimental conditions (Figures 4B,C, respectively), leading to the surprising conclusion that both increased and reduced CaM levels rendered Kv7.2 channels more resistant to PI(4,5)P₂ decrease. Similar results were obtained when untagged Kv7.2 subunits were tested (data not shown).

The role of PI(4,5)P₂ was further evaluated by co-expressing a PI(4)P5-kinase (PIP5K, Figures 4D,E, see also Supplemental Figure 1), which has been shown to increase Kv7.2 current density as a consequence of the tonic elevation of PI(4,5)P₂ levels (Li et al., 2005; Winks et al., 2005; Soldovieri et al., 2016). This effect has been attribute to an increased channel open probability, which is strongly regulated by PI(4,5)P₂ (Li et al., 2005). Consistent with these previous reports, co-expression of PIP5K in HEK293 cells almost doubled Kv7.2 current density (Figure 4D). However, simultaneous expression of PIP5K and CaM resulted in current densities that where smaller than when PIP5K was expressed alone. On the other hand, expression of PIP5K failed to increase Kv7.2 current density when the levels of CaM were reduced by co-expressing the CaM-sponge (Figure 4D). Finally, co-expression of PIP5K resulted in a remarkable resistance to PI(4,5)P₂ depletion, under resting, elevated and reduced CaM levels (Figure 4E), a result consistent with the concept that expression of PIP5K raised PI(4,5)P₂ to concentrations beyond those required to saturate Kv7.2 channels.

Overall, these results show that Ca²⁺-independent CaM-mediated Kv7.2 potentiation depends on PI(4,5)P₂. This can be due to a general effect of CaM on the plasma membrane levels of PI(4,5)P₂, or it might depend on a direct interaction of CaM with Kv7.2. CaM binding to Kv7.2 is improved by CaM phosphorylation which makes the channels more resistant to PI(4,5)P₂ reduction (Kang et al., 2014). CaM phosphorylation is counterbalanced by a resident protein phosphatase 1 (PP1) that binds to the Kv7.2 N-terminus. To unveil if this mechanism is involved in potentiation, we tested the effect of the Kv7.2 N-terminal F24A mutation, which destroys the PP1 binding site (Kang et al., 2014). The results from these experiments revealed that Kv7.2 channels carrying this mutation displayed an increased current density and, importantly, were insensitive to CaM₁₂₃₄ elevation (Figure 5A). Furthermore, F24A channels were less sensitive than wt Kv7.2 to the inhibitory effects of DrVSP activation, and this phenomenon persisted under elevated CaM₁₂₃₄ conditions (Figure 5B).

In many neuronal compartments Kv7.2 heteromerize with Kv7.3 subunits, prompting us to examine whether the observed CaM-induced regulation also occurred in Kv7.2/3 heteromers. In contrast to the effect observed for Kv7.2, CaM (data not show) or CaM₁₂₃₄ co-expression failed to increase the currents carried by Kv7.2/3 channels (Figure 6A).

Elevation of tonic PI(4,5)P₂ levels by PIP5K, alone or in combination with CaM₁₂₃₄, did not result in significant changes in current density from Kv7.2/3 channels (Figure 7A). However, similar to the effect observed on Kv7.2 homomeric channels, overexpression of the CaM sponge to reduce tonic CaM levels led to a decrease in Kv7.2/3 current density that was not counterbalanced by PIP5K co-expression (Figures 6A, 7A, see also Supplementary Figure 1). Instead, the density in the presence of the sponge was significantly reduced when PIP5K was co-expressed (Compare Figures 6A, 7A). Notably, the relationship between conductance and voltage was not significantly altered by manipulations of CaM levels (Figure 6B). Moreover, when compared to homomeric Kv7.2 channels, DrVSP activation caused a much smaller decrease in Kv7.2/3 current size, and either CaM₁₂₃₄ co-expression or depletion (CaM sponge) increased, rather than decreased as in Kv7.2 channels, the effects of DrVSP activation (Figures 7B,C). Thus, incorporating Kv7.3 subunits in heteromeric channels led to a reversal of the response to tonic CaM levels.

To gain further insights into this mechanism, we studied homomeric Kv7.3 channels (Figure 8). To this aim, the A315T Kv7.3 variant, denoted Kv7.3^T, was used because Kv7.3 yields barely detectable currents (Etxeberria et al., 2004; Gómez-Posada et al., 2010). Over-expression of CaM (not shown) or CaM₁₂₃₄ failed to augment current density of Kv7.3^T channels; rather, this perturbation tended to cause a reduction (Figure 8A).

On the other hand, similar to Kv7.2 homomers and Kv7.2/3 heteromers, the density of Kv7.3^T was significantly reduced in the presence of the CaM sponge (Figure 8A), and this reduction was not counterbalanced by the expression of PIP5K (Figure 9); furthermore, PIP5K alone also reduced Kv7.3T current density. Noteworthy, a significant leftward shift in the conductance-voltage relationship of Kv7.3^T channels was observed upon CaM₁₂₃₄ co-expression (Figure 8B). An 84% reduction in Kv7.3^T current density was observed as a consequence of PIP5K expression (Figure 9A), accompanied by a leftward shift of the conductance-voltage relationship (Supplementary Figure 1). Similar results on both current density and gating were observed when homomeric channels formed by either Kv7.3 or Kv7.3T subunits were expressed in CHO cells (data not shown). In addition, Western-blot experiments revealed a clear reduction in the amount of Kv7.3 or Kv7.3^T subunits in both total lysates and plasma membrane-isolated fraction from CHO cells (data not shown). Finally, no significant changes in the sensitivity to acute PI(4,5)P₂ depletion (DrVSP activation) was observed after elevation or reduction of CaM levels (Figures 9B,C).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.