Clancy SM et al. (2009), KCNE1 and KCNE3 beta-subunits regulate membrane...

XB-ART-40120

PLoS One 2009 Jul 22;47:e6330. doi: 10.1371/journal.pone.0006330.

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KCNE1 and KCNE3 beta-subunits regulate membrane surface expression of Kv12.2 K(+) channels in vitro and form a tripartite complex in vivo.

Clancy SM , Chen B , Bertaso F , Mamet J , Jegla T .

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Voltage-gated potassium channels that activate near the neuronal resting membrane potential are important regulators of excitation in the nervous system, but their functional diversity is still not well understood. For instance, Kv12.2 (ELK2, KCNH3) channels are highly expressed in the cerebral cortex and hippocampus, and although they are most likely to contribute to resting potassium conductance, surprisingly little is known about their function or regulation. Here we demonstrate that the auxiliary MinK (KCNE1) and MiRP2 (KCNE3) proteins are important regulators of Kv12.2 channel function. Reduction of endogenous KCNE1 or KCNE3 expression by siRNA silencing, significantly increased macroscopic Kv12.2 currents in Xenopus oocytes by around 4-fold. Interestingly, an almost 9-fold increase in Kv12.2 currents was observed with the dual injection of KCNE1 and KCNE3 siRNA, suggesting an additive effect. Consistent with these findings, over-expression of KCNE1 and/or KCNE3 suppressed Kv12.2 currents. Membrane surface biotinylation assays showed that surface expression of Kv12.2 was significantly increased by KCNE1 and KCNE3 siRNA, whereas total protein expression of Kv12.2 was not affected. KCNE1 and KCNE3 siRNA shifted the voltages for half-maximal activation to more hyperpolarized voltages, indicating that KCNE1 and KCNE3 may also inhibit activation gating of Kv12.2. Native co-immunoprecipitation assays from mouse brain membranes imply that KCNE1 and KCNE3 interact with Kv12.2 simultaneously in vivo, suggesting the existence of novel KCNE1-KCNE3-Kv12.2 channel tripartite complexes. Together these data indicate that KCNE1 and KCNE3 interact directly with Kv12.2 channels to regulate channel membrane trafficking.

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Species referenced: Xenopus
Genes referenced: actl6a ag1 arnt canx kcne1 kcne3 kcne5 kcnh3 kcnh8 kcnj2 mia3

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	Figure 1. Knockdown of endogenous KCNE expression in Xenopus oocytes with siRNA.(A–E) RT-PCR of xKCNE1, xKCNE3 and xKCNE5.1 mRNA isolated from Xenopus oocytes injected with dH2O (−) or the indicated siRNA (+). RT-PCR for β-actin is shown as a control for normalization of band intensity. (A–C) siRNAs designed against xKCNE1, xKCNE3 and xKCNE5.1 all significantly knock down mRNA expression level of their respective target genes. (D–E) In contrast, siRNA targeted to xKCNE1 and xKCNE3 do not affect mRNA expression levels of other KCNE genes. (F) Optical density of cDNA bands relative to β-actin was quantified using NIH ImageJ. xKCNE1, xKCNE3 and xKCNE5.1 siRNA bands indicted a ∼10-fold, ∼4-fold, and ∼4fold reduction in expression of each respective gene; control injections did not show a significant reduction. Each experiment was performed 3 times; values show Mean±SEM, ** p<0.01.
	Figure 2. KCNE1 and KCNE3 inhibit Kv12.2 currents in Xenopus oocytes.(A–E) Current traces recorded from oocytes injected with (A) Kv12.2 cRNA only, or co-injected (B) KCNE1, (C) KCNE3, (D) KCNE1 and KCNE3, or (E) KCNE5.1 siRNAs. Currents were recorded in response to 2 s voltage steps from −100 to +80 mV, in 20 mV increments from a holding potential of −100 mV; tail currents were recorded at −40 mV (protocol in inset). (F) Peak current-voltage relationships from oocytes injected with Kv12.2 cRNA only (▪), or Kv12.2 cRNA plus either KCNE1 (•), KCNE3 (▴), KCNE1 and KCNE3 (▾), or KCNE5.1 (♦) siRNAs. KCNE1 and KCNE3 siRNAs significantly increase Kv12.2 currents and have an additive effect in combination. (Mean±SEM, n = 12–16, p<0.01). (G) Normalized conductance voltage (GV) curves measured from isochronal tail currents for oocytes injected with Kv12.2 cRNA only (▪), and either KCNE1 (•), KCNE3 (▴), or KCNE1+KCNE3 (▾) siRNA. Lines show Boltzmann fits; parameters are given in the Results. Only dual injection of xKCNE1 and xKCNE3 siRNAs caused a significant shift in V50. Data are given as Mean±SEM, n = 12–16, ( p<0.01). (H) Peak current-voltage relationships from oocytes injected with Kv12.2 cRNA (▪), or Kv12.2 cRNA+mKCNE1 (•), mKCNE3 (▴), mKCNE1 and mKCNE3 (▾) cRNA (protocol as in A, Mean±SEM, n = 8, * p<0.05, ** p<0.01). Co-injection of Kv12.2 with mKCNE1 and/or mKCNE3 cRNA significantly reduced Kv12.2 currents. (I) GV curves from isochronal tail currents recorded from oocytes injected with Kv12.2 cRNA (▪), or Kv12.2 and either mKCNE1 (•), mKCNE3 (▴), and mKCNE1 and mKCNE3 (▾) cRNA. Boltzmann fits (lines) revealed that the voltage-dependence of Kv12.2 activation was not significantly affected by overexpression of KCNE1 and KCNE3. V50 values are given in the Results (n = 8).
	Figure 3. Characterization of the Kv12.2 channel antibody.(A) Alignment of a 100 amino acid section, 702–801 of the cytoplasmic C-terminus of mouse Kv12.2 used to generate a Kv12.2-specific polyclonal antibody in rabbit to Kv12.1. The homologous region of Kv12.3 (not shown), is most similar to Kv12.1. Amino acid identities are shaded and numbers indicate amino acid position in Kv12.2. The cartoon depicts a Kv12.2 subunit with 6 transmembrane domains (S1–S6), a Per-Arnt-Sim (PAS) motif and a putative cyclic nucleotide binding motif (cNBD). Gray scale coding indicates the level of amino acid identity shared between Kv12.2 and other Kv12 channels in each region of the channel. (B) Top panel, Western blot analysis demonstrates that the Kv12.2 antibody (anti-Kv12.2) recognizes a single band of ∼120 KD (the predicted size of a Kv12.2 channel monomer) from mouse brain and HEK-293 cells transiently transfected with mKv12.2 channel cDNA (HEK-293+Kv12.2). Anti-Kv12.2 did not recognize specific bands in non-transfected HEK-293 cell lysates (HEK-293), in HEK-293 cells transfected with the closely related Kv12.1 channel (HEK-293+Kv12.1), or when excess antigenic control peptide (CP) was present to block immunodetection (HEK-293+Kv12.2+CP). Bottom panel, anti-β-actin demonstrates equal protein loading for each condition. These experiments were repeated 4 times yielding similar results.
	Figure 4. KCNE1 and KCNE3 reduce membrane surface expression of Kv12.2 channels.(A) Blots of the biotinylated plasma membrane fraction of proteins from Xenopus oocytes injected with Kv12.2 cRNA alone or in combination with KCNE1 and/or KCNE3 siRNA subjected to membrane surface biotinylation. Top panel, Detection of Kv12.2 channel protein with anti-Kv12.2. Xenopus oocytes injected with Kv12.2 cRNA showed just detectable membrane surface expression; whereas the co-injection of KCNE1 and/or KCNE3 siRNA dramatically increased Kv12.2 channel plasma membrane expression. Kv12.2 was not detected in oocytes injected with dH2O (negative control), and was robustly detected in mouse whole brain lysate (positive control). An endoplasmic reticulum (ER) marker (calnexin, middle panel), and a cell membrane marker (β1-integrin, bottom panel), were used as a negative and positive controls, respectively, to show specific isolation of the plasma membrane protein fraction and to confirm equal protein loading. (B) The optical density of each Kv12.2 protein band was quantified using NIH ImageJ and normalized to density of β1-integrin for comparison; both xKCNE1 or xKCNE3 siRNA increased Kv12.2 channel membrane expression>3-fold (Mean±SEM, n = 4, ** p<0.01). Furthermore, the combination KCNE1 and KCNE3 siRNA increased surface Kv12.2 expression ∼20% further than either siRNA alone (n = 4, * p<0.05). (C) Blots showing total protein expression from Xenopus oocytes injected as in A. Top panel, Kv12.2 channel total expression was not detected in dH2O-injected oocytes, and did not increase when Kv12.2 was injected with siRNA for KCNE1 and/or KCNE3. Bottom panel, β-actin was used to confirm equal protein loading for each condition assessed. (D) The optical density of each Kv12.2 protein band was quantified using NIH ImageJ and normalized to β-actin; xKCNE1, xKCNE3 or KCNE1 and KCNE3 siRNA did not significantly affect total Kv12.2 channel expression (Mean±SEM, n = 4).
	Figure 5. Kv12.2 channels simultaneously associate with KCNE1 and KCNE3 β-subunits in vivo.(A) Mouse brain lysates (MB) were immunoprecipitated (IP) with anti-KCNE1, anti-KCNE3, anti-Kv12.2, or anti-Kir2.1 and were then subjected to SDS-PAGE and Western Blot analysis. Whole mouse brain lysate (MB) and proteins precipitated with unconjugated beads (AG only) are shown as positive and negative controls, respectively. Proteins used for IP are indicated at the top, proteins detected in Western blot (IB) are indicated at the left. Top panel, IB with anti-Kv12.2 indicates that both KCNE1 and KCNE3 interact in vivo with Kv12.2 channels. Middle panel, IB was stripped and re-probed with anti-KCNE1. KCNE1 immunoprecipitates with Kv12.2 but not KCNE3. Bottom panel, IB was then re-stripped and re-probed with anti-KCNE3. KCNE3 immunoprecipitates with Kv12.2 but does not interact with KCNE1. Controls show little or no IP of Kv12.2, KCNE1 or KCNE3 with anti-Kir2.1 or unconjugated beads. (B–D) Two-step co-immunoprecipitation assays run under native protein conditions. The Kv12.2 channel complex labeled under native conditions was ∼500 Kd, consistent with a channel tetramer. (B) Kv12.2 channels were immunoprecipitated first with anti-KCNE1 or anti-KCNE3 in these native protein conditions. Specificity was assessed using the anti-Kir2.1 antibody and unconjugated beads as negative controls. Proteins immunoprecipitated in this first IP were subjected to a second IP using anti-Kv12.2. (C) Anti-KCNE1 detected the Kv12.2 tetramer complex after the second IP regardless of whether anti-KCNE1 or anti-KCNE3 was used for the first IP. (D) Similarly, anti-KCNE3 detected the Kv12.2 complex after the second IP with anti-Kv12.2 even if anti-KCNE1 was used for the first IP. These results can be explained if KCNE1 and KCNE3 simultaneously interact with individual Kv12.2 channels. Denatured mouse brain lysate was loaded into the first lane, to confirm the established size of the respective KCNE β-subunit. Note that some KCNE1 has dissociated from the channel complex in the KCNE1 lane of (C).

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