Characterization of the Kv12.2 channel antibody.

<p>(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) <i>Top panel</i>, 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). <i>Bottom panel</i>, anti-β-actin demonstrates equal protein loading for each condition. These experiments were repeated 4 times yielding similar results.</p

KCNE1 and KCNE3 inhibit Kv12.2 currents in <i>Xenopus</i> oocytes.

<p>(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 <i>inset</i>). (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, ** <i>p</i><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 <i>Results</i>. Only dual injection of xKCNE1 and xKCNE3 siRNAs caused a significant shift in V₅₀. Data are given as Mean±SEM, n = 12–16, (** <i>p</i><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, * <i>p</i><0.05, ** <i>p</i><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 <i>Results</i> (n = 8).</p

Knockdown of endogenous KCNE expression in <i>Xenopus</i> oocytes with siRNA.

<p>(A–E) RT-PCR of xKCNE1, xKCNE3 and xKCNE5.1 mRNA isolated from <i>Xenopus</i> 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 <i>NIH ImageJ</i>. 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, ** <i>p</i><0.01.</p

The time course of I_h activation and deactivation is different between ciHCNa and ciHCNb.

<p>A. (Left) Sample current traces elicited by a hyperpolarization of the membrane potential from a holding potential of −10 mV to −90 (ciHCNa) or −70 mV (ciHCNb). The slow portion of the current traces obtained by hyperpolarization to −90 or −70 mV (I_h) was fit with a double (ciHCNa) or single (ciHCNb) exponential function. The sample fits of these traces are shown in dark gray, along with a plot of the residuals of the fits in light gray at the top of each current trace. (Right) Plots of Tau values, which were obtained from fitting with exponential functions, versus test voltage. B. Plot of the ratio of amplitudes of fast component versus the amplitudes of both the fast and slow component of the double exponential fit for the current traces, obtained from oocytes expressing ciHCNa as described in ‘A’, versus test voltage. For both ‘A’ and ‘B’, the values for “n” refer to the number of oocytes used. For all plots, the values represent the mean ± s.e.m.</p

KCNE1 and KCNE3 reduce membrane surface expression of Kv12.2 channels.

<p>(A) Blots of the biotinylated plasma membrane fraction of proteins from <i>Xenopus</i> oocytes injected with Kv12.2 cRNA alone or in combination with KCNE1 and/or KCNE3 siRNA subjected to membrane surface biotinylation. <i>Top panel</i>, Detection of Kv12.2 channel protein with anti-Kv12.2. <i>Xenopus</i> 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, <i>middle panel</i>), and a cell membrane marker (β1-integrin, <i>bottom panel</i>), 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 <i>NIH ImageJ</i> 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, ** <i>p</i><0.01). Furthermore, the combination KCNE1 and KCNE3 siRNA increased surface Kv12.2 expression ∼20% further than either siRNA alone (n = 4, * <i>p</i><0.05). (C) Blots showing total protein expression from <i>Xenopus</i> oocytes injected as in A. <i>Top panel</i>, 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. <i>Bottom panel</i>, 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 <i>NIH ImageJ</i> 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).</p

Phylogenetic pattern and N-linked glycosylation of a subset of <i>Ciona</i> HCNs.

<p>A. An alignment of the pore region produced by ClustalX and generated by GeneDoc. Red boxes indicates known and putative HCN N-linked glycosylation site and the yellow line indicates the division between the presence and absence of this functional site. B. An HCN cladogram generated by aligning the sequences shown as described in the text. “X” indicates the predicted emergence point of N-linked glycosylation in the HCN family. Sequences other than those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047590#pone-0047590-g001" target="_blank">Figure 1</a>, and other than those for <i>Oikopleura dioica</i> and <i>Branchiostoma floridae</i> (lancelet), are: gi: 260829977; <i>Strongylocentrotus purpuratus</i> (sea urchin) gi:47551101; <i>Panulirus interruptus</i>, splice variant I (lobster) gi:115334851; <i>Apis mellifera</i> (bee) gi:33355927; <i>Drosophila melanogaster</i>, isoform B, gi:84795752; <i>Aedes aegypti</i> (mosquito) gi:108875949. C. Western blot of membrane fractions from oocytes injected with cRNA of ciHCNa, ciHCNa-N380Q or ciHCNb, untreated (−) or treated (+) with PNGaseF and probed with anti-V5 antibody. Actin, robed with an anti-actin antibody, was used as a loading control. ciHCNa, but not ciHCNa-N380Q or ciHCNb, is shifted to a lower molecular mass in the presence of PNGaseF. Molecular weights, indicated by the short black bars to the right of the blots, are 130 kDa (top), 100 kDa (upper middle) and 70 kDa (lower middle) and 35 kDa (bottom). D. Western blot of whole cell lysates from CHO cells transfected with 1.5 ug cDNA of ciHCNa, ciHCNa-N380Q or ciHCNb, either treated (+) or untreated (−) with PNGaseF and probed for using an anti-V5 antibody. Molecular weights, indicated by the short black bars to the right of the blots, are 95 kDa (top), 72 kDa (middle) and 34 kDa (bottom). Predicted mass of <i>Ciona</i> HCN channels without post-translational modification, is 78.8 kDa and 93.3 kDa for ciHCNa and ciHCNb, respectively.</p

Kv12.2 channels simultaneously associate with KCNE1 and KCNE3 β-subunits <i>in vivo</i>.

<p>(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. <i>Top panel</i>, IB with anti-Kv12.2 indicates that both KCNE1 and KCNE3 interact <i>in vivo</i> with Kv12.2 channels. <i>Middle panel</i>, IB was stripped and re-probed with anti-KCNE1. KCNE1 immunoprecipitates with Kv12.2 but not KCNE3. <i>Bottom panel</i>, 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).</p

RT-PCR Xenopus (x) primer sequences.

<p>RT-PCR Xenopus (x) primer sequences.</p

siRNA KCNE gene sequences.

<p>siRNA KCNE gene sequences.</p

Sodium passes through both <i>Ciona</i> HCNs but does not enhance current flowing through them.

<p>A. Plots of instantaneous I_h versus voltage at the indicated concentrations of extracellular cations. Values were determined from current traces elicited using the same protocol as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047590#pone-0047590-g005" target="_blank">Fig. 5</a> and represent mean ± s.e.m. for each test voltage; black line/black squares for 100 mM K⁺, light gray line/black circles for 80 mM Na+/20 mM K+, black line/black upward triangles for 80 mM Li/20 mM K+, black line/black downward triangles for 20 mM K+. Lines represent linear fits through the mean values. B. Bar graphs showing the reversal potentials and slope conductance for each <i>Ciona</i> HCN, determined from the plots in ‘B’ where values from individual experiments were fit by a straight line through the linear portion of the curve across the x-axis. The number of oocytes that were used in each group is shown below each bar in brackets; reversal potential and slope conductance were obtained from the same current voltage data. Values represent mean ± s.e.m. for solution used, which were compared using a one-way ANOVA and tukey's post test. * indicates a difference (p<0.05) when compared to all other conditions. ** indicates a difference (p<0.05) when compared to * or ***. *** indicates a difference (p<0.05) when compared to ** or *.</p