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. 2000 May 9;97(10):5562-7.
doi: 10.1073/pnas.100118597.

A neuronal beta subunit (KCNMB4) makes the large conductance, voltage- and Ca2+-activated K+ channel resistant to charybdotoxin and iberiotoxin

P Meera  1 , M Wallner, L Toro
Affiliations

A neuronal beta subunit (KCNMB4) makes the large conductance, voltage- and Ca2+-activated K+ channel resistant to charybdotoxin and iberiotoxin

P Meera et al. Proc Natl Acad Sci U S A. .

Abstract

Large conductance voltage and Ca(2+)-activated K(+) (MaxiK) channels couple intracellular Ca(2+) with cellular excitability. They are composed of a pore-forming alpha subunit and modulatory beta subunits. The pore blockers charybdotoxin (CTx) and iberiotoxin (IbTx), at nanomolar concentrations, have been invaluable in unraveling MaxiK channel physiological role in vertebrates. However in mammalian brain, CTx-insensitive MaxiK channels have been described [Reinhart, P. H., Chung, S. & Levitan, I. B. (1989) Neuron 2, 1031-1041], but their molecular basis is unknown. Here we report a human MaxiK channel beta-subunit (beta4), highly expressed in brain, which renders the MaxiK channel alpha-subunit resistant to nanomolar concentrations of CTx and IbTx. The resistance of MaxiK channel to toxin block, a phenotype conferred by the beta4 extracellular loop, results from a dramatic ( approximately 1,000 fold) slowdown of the toxin association. However once bound, the toxin block is apparently irreversible. Thus, unusually high toxin concentrations and long exposure times are necessary to determine the role of "CTx/IbTx-insensitive" MaxiK channels formed by alpha + beta4 subunits.

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Figures

Figure 1
Figure 1
Molecular properties of the MaxiK channel β3 and β4 subunits. (a) Multiple sequence alignment of four human MaxiK channel β subunits: β1 (KCNMB1) (8), β2 (KCNMB2) (9), β3 (KCNMB3) (28), and β4 (KCNMB4) (GenBank accession nos. β1, U25138; β2, AF099137; β3, AF160968; and β4, AF160967). Con, consensus sequences; TM1 and TM2, transmembrane regions (shaded boxes). Boxes show consensus sequences for protein kinase A phosphorylation (K/R,K/R,X1–2,S/T), N-linked glycosylation (N,X,S/T), and conserved cysteines. (b) Dendrogram of human β subunits. Length of vertical branches is inversely proportional to the similarity among sequences. The sequence similarity among the four human β subunits ranges from 36 to 54%. (c) In vitro translation and deglycosylation of β1–β4 mRNA. Transcripts were in vitro translated in a reticulocyte lysate in presence of microsomal membranes (Promega) and [35S]methionine. Microsomal proteins were treated with (+) or without N-glycosidase F (NEB) (−) and resolved by SDS/PAGE. Molecular weights were calculated with the gcg program; for each N-linked glycosylation site, 4 kDa was added to the molecular mass (calc. MM). Standards are in kilodaltons (kDa).
Figure 2
Figure 2
β4 mRNA is abundant in brain. (a) PolyA+ RNA Dot blot (CLONTECH) of 50 human tissues were hybridized at high stringency with a 32P-labeled β4 (681 bp, AccI–AccI fragment) probe. Tissues are: whole brain (1A), amygdala (2A), caudate nucleus (3A), cerebellum (4A), cerebral cortex (5A), frontal lobe (6A), hippocampus (7A), medulla oblongata (8A), occipital lobe (1B), putamen (2B), substantia nigra (3B), temporal lobe (4B), thalamus (5B), nucleus accumbens (6B), spinal cord (7B), heart (1C), aorta (2C), skeletal muscle (3C), colon (4C), bladder (5C), uterus (6C), prostate (7C), stomach (8C), testis (1D), ovary (2D), pancreas (3D), pituitary gland (4D), adrenal gland (5D), thyroid gland (6D), salivary gland (7D), mammary gland (8D), kidney (1E), liver (2E), small intestine (3E), spleen (4E), thymus (5E), peripheral leukocyte (6E), lymph node (7E), bone marrow (8E), appendix (1F), lung (2F), trachea (3F), placenta (4F), no sample (5F-8F, G8), fetal brain (G1), fetal heart (G2), fetal kidney (G3), fetal liver (G4), fetal spleen (G5), fetal thymus (G6), fetal lung (G7), 100 ng yeast total RNA (H1), 100 ng of yeast tRNA (H2), 100 ng of E. coli rRNA (H3), 100 ng of E. coli DNA (H4), 100 ng of poly r(A) (H5), 100 ng of human Cot 1 DNA (H6), 100 ng of human DNA (H7), and 500 ng of human DNA (H8). The amount of RNA in each dot is between 89 and 514 ng, due to a normalization with respect to ubiquitously expressed mRNAs. (Inset) Internal dot blot standard of β1, β2, and β4 transcripts. The internal standard shows a detection limit of 0.01 fmol or 6 × 106 transcripts. (b) Quantification of Dot blot signals using phosphorimaging. The signals were background subtracted and normalized to equal amounts of RNA. Absolute transcript numbers were obtained using the signals from the internal standard assuming similar hybridization efficiencies. (c) Northern Blots were hybridized and visualized as in a. The signals on the blot with fetal tissues are stronger than for the blot with adult tissues (c); however, for a comparison of expression levels, compare signals on the same blot (a and b). (d) β4 gene is localized in chromosome 12q15–21. Double labeling of chromosome 12 with a chromosome 12 centromere-specific probe (red signal) and genomic β4 probe (green signal) (n = 10 measurements).
Figure 3
Figure 3
CTx and IbTx resistant MaxiK channel formed by α + β4 subunits. Outside-out currents in 110 mM symmetrical K+ (100 μM Ca2+ in the pipette) from ooyctes expressing α + β4, α + β1 and α subunits. The holding potential was 0 mV. Pulses were to + 40 mV for 40 ms with 2-s interpulse interval. Channels with an open probability near 1 were examined. Currents were measured 3–5 min after toxin application. (a and b) Traces are normalized (to control currents before toxin application) currents after application of 100 nM CTx (a) or 100 nM IbTx (b). (Inset) Topology of β1 and β4 subunits. (c and d) Time course for CTx (c) and IbTx (d) block of α + β4 subunits. Arrows mark the time of toxin addition. (Insets) Original current traces. To illustrate the dramatic difference between α and α + β4 toxin blockade, we calculated the time course for the α subunit blockade using the CTx and IbTx average kon and koff values (see Fig. 4).
Figure 4
Figure 4
β4 subunit dramatically slows down CTx and IbTx association and dissociation. Currents in outside-out patches were evoked by repetitive pulses as in Fig. 3. Currents were measured at the end of the pulse and plotted as a function of time; arrows mark the time of toxin application and washout. To attain toxin block in a reasonable time, CTx concentrations were 3 nM for α (a) and α + β1 (b), and 500 nM for α + β4 (c). IbTx concentrations were 10 nM for α (d), 100 nM for α + β1 (e), and 1 μM for α + β4 (f). kon, koff, and Kd values are given in M−1s−1, s−1, and nM, respectively, and were obtained as described in Experimental Procedures. Gray lines are fits to data. (g) Mean kon values (M−1s−1) ± SD (error bars) for CTx and IbTx block in α, α + β1, and α + β4 channels. CTx: kon-α = 2 ± 1 × 107 (n = 7), kon-α+β1 = 1.9 ± 1.8 × 106 (n = 4), and kon-α+β4 = 8 ± 7 × 104 (n = 10); IbTx: kon-α = 2 ± 0.7 × 106 (n = 4), kon-α+β1 = 1.1 ± 0.5 × 105 (n = 3), and kon-α+β4 = 4 ± 2 × 103 (n = 4). n = number of experiments. For both toxins, values for α + β1 and α + β4 were significantly different from those of α subunit (P < 0.005). In two out of seven cases, IbTx block of α + β4 channels had even slower on-rates (not included in mean value).
Figure 5
Figure 5
The extracellular loops of β4 and β1 subunit determine the kinetics of toxin block. Experiments were performed as in Fig. 4. (a) 100 nM CTx was applied to α + β1Lβ4 channels (β1 carrying the loop of β4) and 3 nM CTx (b) to α + β4Lβ1 channels (β4 carrying the loop of β1). Mean kon values (M−1s−1) ± SD were: for α + β1Lβ4, 4 ± 2 × 104 (n = 3); for α + β4Lβ1, 7 ± 2 × 106 (n = 4). Mean koff values (s−1) were for α + β4Lβ1, 1 ± 0.2 × 10−2. (c) Model of channels formed by α + β subunits where the β-subunit extracellular loop may contribute to the toxin (Tx) receptor. Two of the four subunits are shown.
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