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PLoS One
2011 Jan 01;610:e26524. doi: 10.1371/journal.pone.0026524.
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Four and a half LIM protein 1C (FHL1C): a binding partner for voltage-gated potassium channel K(v1.5).
Poparic I
,
Schreibmayer W
,
Schoser B
,
Desoye G
,
Gorischek A
,
Miedl H
,
Hochmeister S
,
Binder J
,
Quasthoff S
,
Wagner K
,
Windpassinger C
,
Malle E
.
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Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G(0)/G(1) phase. Furthermore, low expression of K(v1.5), a voltage-gated potassium channel known to alter myoblast proliferation during the G(1) phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between K(v1.5) and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and K(v1.5) within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of K(v1.5) with FHL1C in Xenopus laevis oocytes markedly reduced K(+) currents when compared to oocytes expressing K(v1.5) only. We here present the first evidence on a biological relevance of FHL1C.
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22053194
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Figure 1. Wild-type FHL1 protein isoforms and mutated FHL1A proteins.Top: FHL1A, FHL1B and FHL1C protein structures (resulting from alternative splicing of the wild-type FHL1 gene), including the LIM domain architecture and functional domains (RBP-Jk, recombination signal-binding protein 1 for J-kappa; NLS, nuclear localization signals; NES, nuclear export sequence) are shown for the corresponding variants. Bottom: Protein structures resulting from alternative splicing of the two different mutant FHL1 genes. An arrow in the fourth LIM domain of mutated FHL1A at position 224 (termed FHL1Ap.C224W) indicates the position of the amino acid exchange. The dotted line suggests alterations in the LIM-like architecture (termed MUT1 here). FHL1Ap.G168fs is identical to FHL1C protein (termed MUT2 here) (Gâ=âglycine, fsâ=âframe shift).
Figure 2. Proliferation rate of human myoblasts.Myoblasts from controls (WT1, WT2) and patients (MUT1, MUT2) were cultured for the indicated times (A) or for 48 h (B). Myoblast samples WT1 and MUT1 originated from tibialial anterior muscle biopsy, samples WT2 and MUT2 from biceps brachii muscle biopsy. (A) Cell proliferation was determined by measuring the number of viable cells using Casy cell counter. Values represent mean ± SD of three experiments (six wells per one experiment). **p<0.01 and *** p<0.001. (B) Flow cytometric measurements (see Methods) were performed to estimate cell cycle, i.e. G0/G1 phase, S phase, and M phase, respectively. One representative experiment (performed in triplicate) out of three is shown.
Figure 3. RT-PCR and Western blot for FHL1 and Kv1.5 in human myoblasts.(A) RNA was isolated from myoblasts from controls (WT1, WT2) and XMPMA patients (MUT1, MUT2) and the corresponding Kv1.5 and FHL1 regions were amplified by RT-PCR. Primers, spanning from exon 1 to 2 (termed FHL1) amplify the mRNA stretch coding for the common N-terminus of FHL1 in all three FHL1 isoforms (FHL1A, FHL1B and FHL1C), while primers termed FHL1A or FHL1C specifically amplify mRNAs encoding FHL1A or FHL1C, respectively. (Pâ=âpositive control: human fetal brain marathon cDNA). To ensure equal gel loading, RT-PCR for human HPRT1 was performed. One representative experiment out of three is shown. See Table S1 for the primer sequences used. (B) Protein lysates from myoblasts from controls (WT1, WT2) and XMPMA patients (MUT1, MUT2) were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes and immunoreactive bands were detected with anti-Kv1.5 or anti-FHL1A as primary antibodies. After stripping, the membranes were incubated with anti-β-actin antibody. (Kv1.5â=â68 kDa; FHL1Aâ=â32 kDa; β-actinâ=â45 kDa). One representative experiment out of three is shown.
Figure 4. Subcellular localization of heterologous expressed chimaeric FHL1C and Kv1.5 constructs in the atrial HL-1 cell line.Cells were cultured as described and after transfection, fluorescence of either eYFP- and eCFP-labelled proteins was detected as described in the Methods section. Each horizontal sequence of images was taken from the same cell and vertical section. For better visualization of colocalization, the eYFP and eCFP channels are shown in green and red, respectively, hence yellow in the overlay image indicates colocalization (merge). Intensity histograms are shown to quantify colocalization of both markers (right). Histogram shows the pixel-by-pixel analysis of the section indicated by the yellow line in the merged channel (from left to right). (A) Staining for FHL1CeYFP and Kv1.5eCFP and colocalization. (B) Staining for Kv1.5eYFP (upper) and FHL1CeYFP (lower) and colocalization with SrÃeCFP (a marker for the endoplasmic reticulum). (C) Staining for Kv1.5eYFP (upper) and FHL1CeYFP (lower) and colocalization with GPIeCFP (a marker for lipid raft domains of the plasma membrane). One representative series of images out of three independent series of experiments is shown.
Figure 5. Physical interaction of FHL1C with the Kv1.5 cytosolic C-terminus.(A) Autoradiography of the dried gel, showing the amount of radioactive protein associated with the bait. As radioactive protein either FHL1C or the Ã/γ subunits of heterotrimeric G-protein (GÃ/γ; control) was used as indicated at the bottom of the lanes. (B) Commassie Brilliant Blue staining of the SDS gel shown in panel A. Bait proteins used are indicated at the bottom of the lanes: GST protein alone (GST); GST-Kv1.5 C-terminus fusion protein (Kv1.5); C-terminus of G-protein activated inwardly rectifying potassium channel (GIRK1) fused with GST (G1-CT) as positive control. (C) Statistics of the bound radioactivity normalized to the amount of bait protein (relative specific radioactivity) for FHL1C using Kv1.5 and GST as a bait (left) and GÃ/γ using G1-CT and GST as a bait (right). Values represent the mean values from different experiments (N is given in parenthesis above each bar). Significant difference (**p<0.01) between GST and Kv1.5 /G1-CT.
Figure 6. Coexpession of FHL1C reduces currents through Kv1.5 channels in Xenopus laevis oocytes.(A) Representative original current traces recorded from two oocytes expressing either Kv1.5 alone (upper panel) or Kv1.5 in combination with FHL1C (lower panel). Each family of current traces originated from four different suprathreshold voltage pulses (â35, â10, +10 and +40 mV). Recordings were taken five days after cRNA injection. (B) Ipeak/Em relation for the oocytes shown in (A). Original data (circles) and fit through the data according to a Boltzmann isotherm (solid lines) are shown. (C) Representative statistics of steady-state activation (nâ) five days after cRNA injection (with and w/o 500 ng cRNA encoding FHL1C coexpressed). Circles represent mean values ± SEM from six batches of oocytes (five oocytes per batch and experimental condition) and the dotted lines fits trough the data according to Boltzmann isotherms, respectively. (D) Kv1.5 inactivation kinetics, same data set as in C. Mean values of the current inactivation ratio () ± SEM are shown. (E) Time course of the effect of FHL1C coexpression on maximal K+ conductance. Data were normalized to Gmax of oocytes expressing Kv1.5 alone for five days (mean values ± SEM of six different batches of oocytes are shown); at least five oocytes were recorded from every batch and experimental group. (F) Dose dependent effect of coexpression of FHL1C on Gmax; five days after cRNA injection. The amount of cRNA coinjected, encoding FHL1C, is shown at the bottom. Mean values ± SEM from six different experiments (five oocytes per batch and experimental group) is shown (ns: the difference is statistically not significant, *: p<0.05 and ***: p<0.001, respectively).
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