Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 6 (9), e25508

Kv7 Channels Can Function Without Constitutive Calmodulin Tethering

Affiliations

Kv7 Channels Can Function Without Constitutive Calmodulin Tethering

Juan Camilo Gómez-Posada et al. PLoS One.

Abstract

M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The S511D and I340E mutations disrupt the CaM interaction.
A. Emission spectra of 12.5 nM D-CaM in the absence (left panel; 10 mM EGTA and no added Ca2+), and in the presence of 2.0 µM free Ca2+ (right panel), as well as in the presence of molar excess of the indicated GST-Kv7.2-CaMBD fusion proteins (200 nM). Note the difference in the fluorescence emission axis, as well as the shift in the peak emission to the left in the presence of Ca2+. The emission spectra of D-CaM alone and in the presence of I340E or S511D were indistinguishable. B. Relative fluorescence increase of D-CaM. Bars represent the mean ± SEM of the percentage increase in fluorescence emission (n≥4). C. Co-immunoprecipitation of full-length Myc-tagged Kv7.2 and CaM. Proteins from HEK293T cells expressing the constructs indicated were immunoprecipitated with the anti-Myc antibody, separated by SDS-PAGE and analyzed in Western blots probed with anti-Myc or anti-CaM antibodies (n = 4). D. Co-immunoprecipitation of N-terminal HA-tagged Kv7.3 assembled with N-terminal Myc-tagged Kv7.2. Western blots were probed with anti-Myc, anti-HA or anti-CaM antibodies (n = 4).
Figure 2
Figure 2. The CaM-binding incompetent S511D mutant is functional, whereas the CaM-binding incompetent I340E mutant is not.
A. Left column, schematic illustration of the CaM binding region. Boxes represent helices A and B. Middle column, representative current traces from Xenopus oocytes expressing homomeric Kv7.2 WT, I340E or S511D channels. Currents were elicited by 800 ms jumps to potentials between -120 and +50 mV from a holding potential of -50 mV. Tail currents were measured at -20 mV. Right column, the difference in amplitude of the relaxation measured at -20 mV after a pulse to -120 mV and to +50 mV was measured. Bars show the mean relaxation amplitudes measured at -20 mV for oocytes expressing the indicated homomeric channels (n≥10). The difference in current between WT and S511D was not significant (Unpaired Student's t test). B. Representative current recordings from Xenopus oocytes injected in a 1∶1 ratio of cRNAs for Kv7.3, and Kv7.2, Kv7.2-I340E or Kv7.2-S511D. Right column, normalized average conductance. Bars represent the mean ± SEM of the normalized conductance of Kv7.2/3 heteromeric channels (n≥5). C. Normalized surface expression of Kv7.3 subunits tagged with HA in the extracellular S1-S2 loop after co-expression with Kv7.2, Kv7.2-I340E or Kv7.2-S511D. Xenopus oocytes were injected with a 1∶1 ratio of cRNAs for Kv7.3 and Kv7.2 (n = 12, two batches). The background of uninjected oocytes was subtracted and the values given are the means (± SEM) normalized to the values obtained from WT-Kv7.2/3 channels from the same batch. The difference in surface expression was not statistically significant (Unpaired Student's t test).
Figure 3
Figure 3. The S511D mutation did not restore the function of other CaM binding perturbing mutations.
Left column, schematic illustration of the CaM binding region as in Fig. 2. Kv7.2 subunits are represented by white boxes and Kv7.3 subunits by black boxes. Crosses indicate point mutations known to prevent CaM binding to Kv7.2 introduced at equivalent position in Kv7.2 or Kv7.3. Middle column, representative current recordings from Xenopus oocytes injected in a 1∶1 ratio of cRNAs for Kv7.3, Kv7.2, or the different mutants studied. Right column, normalized average conductance. Bars represent the mean ± SEM of the normalized conductance mutant Kv7.2/3 channels (n≥4). The dotted line is the reference normalized conductance from K7.2/3. A. The double mutant I340E/S511D Kv7.2 co-expressed with WT Kv7.3 was not functional. B. Introduction of CaM binding perturbing mutations in helix A or helix B of Kv7.3 lead to non-functional channels. Left, schematic representation of the mutants analyzed. Middle column, representative current traces of whole-cell patch-clamp recordings from HEK293T expressing homomeric Kv7.3T WT, I379A or A518D. The pore A315T mutation was introduced to boost expression -. C and D. Kv7.2 S511D or WT did not restore the function of Kv7.3 subunits carrying mutations in helix A or helix B. Currents were elicited with 800 ms jumps to potentials between -100 and +60 mV from a holding potential of -20 mV. Tail currents were measured at -20 mV. Right column, the difference in the amplitude of the relaxation measured at -20 mV after a pulse to -100 mV and +60 mV was measured (n≥8).
Figure 4
Figure 4. The double mutation I340E/S511D impairs plasma membrane expression.
A. Left, flow cytometry histogram of cell surface staining. Center, schematic representation of the AB mutants analyzed. Right, the normalized sum of the product of the number of events and fluorescence intensity from the flow cytometry histogram distribution is plotted as surface expression index. Dotted lines are the mean values of AB-I340E and AB-WT. There was no significant difference in the surface expression index of the S511D and WT construct, but the index was significantly different to that of I340E (n = 4). B. The glycosylation of the I340E/S511D mutant was larger than that of the I340E and not significantly different to that of the S511D mutant. Western blot probed with an anti-GFP antibody of HEK293T cell extracts expressing Tac-AB constructs carrying the indicated mutations. The bands represent immature (#) and mature (##) forms of the Tac chimeras. Right, averaged ratio of the optical density (mature OD/(immature OD + mature OD)) normalized to the Tac-AB ratio (n≥3). ***, significance at P≤0.001, ** P≤0.01, paired Student's t test. C. Confocal images of HEK293T cells transfected with WT and mutant channels tagged with an HA extracellular epitope and mCFP. A deletion between helix A and B was introduced to boost surface expression (see text). HEK293T cells transfected with the indicated cDNAs were surface stained with anti-HA antibodies. The proportion of the cells with surface staining was determined in > 40 CFP positive cells for each construct in three independent experiments. Grey bars represent mean ± SEM of the fluorescence intensities per unit area obtained for surface (red channel) divided by total fluorescence. Fewer cells expressed S511D at the surface (black bars), and in those cells, the average surface expression was reduced (grey bars). * P≤0.05; *** P≤0.001; unpaired Student's t test.
Figure 5
Figure 5. Activation of Ca2+-CaM does not inhibit Kv7.2/3 in Xenopus oocytes.
A. Representative time-course of M1-muscarinic receptor activation by carbachol when expressed in oocytes co-expressing Ca2+-CaM regulated hEag-1 potassium channels (n = 4). The normalized instantaneous current from −80 mV to −0 mV is plotted as white circles and corresponds to the activation of the endogenous Ca2+-dependent Cl- current. The size of the post-pulse relaxation at 0 mV is plotted as black circles and corresponds to the opening of hEag-1 channels. The activation of the Cl- current demonstrated that intracellular Ca2+ increased, and the inhibition of the hEag1 currents demonstrated that oocytes support Ca2+-CaM-mediated inhibition of potassium channels. B. Representative traces from A before and during the application of 100 µM carbachol. C. Representative time-course of M1-muscarinic receptor activation when expressed in oocytes co-expressing Kv7.2/3 potassium channels (n = 6). Same layout as in A. The size of the post-pulse relaxation at 0 mV is plotted as black circles and corresponds to the opening of Kv7.2/3 channels. D. Representative traces from C before and during the application of 100 µM carbachol.
Figure 6
Figure 6. Effect of CaM overexpression on maximal conductance.
Bars represent the mean relative reduction in maximal conductance co-expressing Kv7.3 with either WT or S511D subunits after overexpression of CaM in Xenopus oocytes. The maximal conductance was obtained after fitting a Boltzmann distribution to the current-voltage relationships from tail currents measured at −20 mV. Currents were elicited by 800 ms jumps to potentials between −120 and +50 mV from a holding potential of -50 mV. There was a significant difference between the two groups (*** P<0.0001, unpaired Student's t test: n≥6; two batches of oocytes).

Similar articles

See all similar articles

Cited by 13 PubMed Central articles

See all "Cited by" articles

References

    1. Schroeder BC, Kubisch C, Stein V, Jentsch TJ. Moderate loss of function of cyclic-AMP-modulated KCNQ2/KCNQ3 K+ channels causes epilepsy. Nature. 1998;396:687–690. - PubMed
    1. Maljevic S, Wuttke TV, Seebohm G, Lerche H. Kv7 channelopathies. Pflugers Arch. 2010;460:277–288. - PubMed
    1. Yus-Nájera E, Santana-Castro I, Villarroel A. The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels. J Biol Chem. 2002;277:28545–28553. - PubMed
    1. Haitin Y, Attali B. The C-terminus of Kv7 channels: a multifunctional module. J Physiol. 2008;3:1803–1810. - PMC - PubMed
    1. Selyanko AA, Brown DA. Intracellular calcium directly inhibits potassium M channels in excised membrane patches from rat sympathetic neurons. Neuron. 1996;16:151–162. - PubMed

Publication types

Feedback