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Fig. 1.
Xenopus Kvβ subunits. The predicted amino acid sequences of Xenopus xKvβ2 and xKvβ4 coding regions are aligned against mammalian Kvβ1, Kvβ2, and Kvβ3 sequences [hKvβ1.1 and hKvβ2.1 (McCormack et al., 1995); hKvβ3.1 (Leicher et al., 1998)]. Gaps (dots) have been introduced to improve the alignment. Residues that are identical to those of xKvβ2 are indicated by a hyphen. Overall, xKvβ2 and xKvβ4 share 71% identity at the amino acid level (Table 1). α-Helices and β-sheets are shadedlight and dark gray, respectively; residues that contribute to the putative active site are indicated by an overlying asterisk (Gulbis et al., 1999).
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Fig. 2.
xKvβ2 and xKvβ4 transcripts are present in excitable tissues of developing Xenopus embryos. Embryos were hybridized as whole mounts to antisense or sense cRNA probes corresponding to either xKvβ2 (A–F) or xKvβ4 (G–I). The in situ hybridization signal is recognizable as a deep blue-purple precipitate. The whole-mount embryos (A–C, G–I) are oriented with their dorsal sides up and anterior ends to theleft. Embryos that were hybridized to sense control probes are identified by s; these demonstrate the background signal (caused by trapping of probe in internal cavities), which is higher in older embryos. The transverse sections(D–F) are oriented dorsal sideup. A, At 26 hr (St 24), xKvβ2 mRNA is prominent in developing somites. B, Between 1 and 2 d (top, middle, and bottom embryos are St 28, 29, and 30, respectively), the pattern of expression of xKvβ2 mRNA undergoes a dramatic change. Initially the signal predominates in the somites but with time diminishes in this tissue and begins to appear in the spinal cord (arrowheads). At St 35–36 (50 hr; C), the signal is no longer observed in the somites but is present in the spinal cord and head. D, In a transverse section through a 1 d embryo hybridized to xKvβ2 antisense probe, the signal is present in the developing somites. E, In a transverse section of a 2 d embryo hybridized to xKvβ2 antisense probe, the signal is detectable in the dorsal spinal cord, where Rohon-Beard cells reside. F, In a transverse section of a 2 d embryo hybridized to a sense control probe, no signal is present.G–I, Whole-mount embryos hybridized to xKvβ4 antisense probe. At all stages, the signal predominates in the nervous system. In the older embryos (e.g., I), the signal is stronger. Comparison of H andI (xKvβ4) with B and C(xKvβ2) reveals that xKvβ4 mRNA localizes to a more ventral position in the spinal cord than does xKvβ2. Scale bar:A, G, 1 mm; B,C, 1.5 mm; H, 1.2 mm;I, 1.1 mm; D–F, 700 μm.
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Fig. 3.
Coexpression of xKvβ2, but not xKvβ4, with xKv1.1α or xKv1.2α subunits leads to an increase in current amplitude. A, Representative whole-oocyte recordings of currents induced after injection of Kv1.1α (top) and Kv1.2α (bottom) RNAs in the absence (left) or presence of xKvβ2 (middle) or xKvβ4 RNA (right). Currents were elicited by depolarizing the membrane in 10 mV increments to potentials ranging between −60 and +130 mV from a holding potential of −80 mV; currents elicited by depolarizations to −60, −30, 0, +30, +60, +90, and + 120 mV are shown. B, Normalized current–voltage (I–V) relationships for currents recorded from oocytes injected with xKv1.1α (left) and Kv1.2α (right) ±xKvβ2 or xKvβ4 RNAs. Current amplitudes were normalized to the mean current amplitude obtained at +50 mV for xKv1.1α or Kv1.2α alone; numbers in parentheses =n. Data obtained in the presence of Kvβ2 (▪) are significantly different from those obtained in its absence (○);p ≤ 0.0002.
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Fig. 4.
Coexpression of xKvβ2 versus xKvβ4 subunits differentially modulates steady-state activation properties of xKv1α channels. A, B, Conductance–voltage (G–V) relationships indicate that coexpression of either xKv1α subunit (A, xKv1.1α;B, xKv1.2α) with xKvβ2 subunits results in an increase in G max. In contrast, coexpression of xKv1α subunits with xKvβ4 has no significant effect onG max. C, D, NormalizedG–V curves for xKv1.1α (C) or xKv1.2α (D) currents obtained in the absence or presence of xKvβ subunits. Coexpression of xKv1.2α with either xKvβ2 or xKvβ4 subunits leads to a depolarizing shift in the G–Vrelationship (Table 2). In C, data obtained in the presence of xKvβ4 (▴) are statistically different from those obtained in its absence (○) in the range of −20 to +40 mV;p values range between 0.0001 and 0.006. InD, data obtained in the presence of xKvβ2 (▪) or xKvβ4 (▴) are statistically different from those obtained in its absence (○) in the range of +10 to +50 mV (xKvβ2) and 0 and 50 mV (xKvβ4); p values range between 0.0001 and 0.04.
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Fig. 5.
Coexpression of xKvβ with xKv1α subunits accelerates kinetics of activation. Time to half-maximum activation (t 1/2) as a function of voltage is plotted for recordings obtained from whole oocytes injected with Kv1.1α (left) or Kv1.2α (right) cRNAs in the absence and presence of xKvβ2 or xKvβ4 cRNAs. Representative currents are presented in Figure 3. Data obtained in the presence of either xKvβ are significantly different from those obtained in its absence; p values are all <0.0001. The number of oocytes recorded from are as follows: Kv1.1α alone, 72; Kv1.1α + xKvβ2, 70; Kv1.1α + xKvβ4, 35; Kv1.2α alone, 83; Kv1.2α + xKvβ2, 82; Kv1.2α + xKvβ4, 49.
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Fig. 6.
Activation of xKv1α currents recorded in oocyte macropatches is accelerated when xKvβ subunits are coexpressed. Representative macropatch recordings (A) and time to half-activation (t 1/2)–voltage relations (B, C) are presented for currents recorded from oocytes injected with xKv1.1α (A,left; B) or xKv1.2α (A,right; C) coexpressed with xKvβ subunits. Currents were elicited by depolarizing the membrane in 10 mV increments to potentials ranging between −60 and +130 mV from a holding potential of −80 mV; the currents elicited by depolarizations to −60, −30, 0, +30, +60, +90, and +120 mV are shown. InB, data obtained in the presence of Kvβ2 are significantly different from those obtained in its absence in the range of +10 to + 60 mV, except at +40 mV; p values range between 0.02 and 0.05. Data obtained in the presence of Kvβ4 are significantly different from those obtained in its absence in the range of −30 to +70 mV; p values range between 0.0005 and 0.04. In C, data obtained in the presence of Kvβ2 are significantly different from those obtained in its absence in the range of −30 to +60 mV; p values range between 0.0006 and 0.02. Data obtained in the presence of Kvβ4 are significantly different from those obtained in its absence in the range of +10 to +60 mV; p values range between 0.02 and 0.04.
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Fig. 7.
Coexpression of xKvβ4 induces inactivation of both xKv1.1α and xKv1.2α currents. A, Coexpression of xKvβ4, but not xKvβ2, induces inactivation of xKv1α channels (*p ≤ 0.0001 vs Kv1α alone). B, For both xKv1.1α and xKv1.2α subunits, coexpression of xKvβ4 induces a voltage-dependent inactivation; more inactivation is observed at stronger depolarizations.
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kcnab2 (potassium channel, voltage gated subfamily A regulatory beta subunit 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
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kcnab2 (potassium channel, voltage gated subfamily A regulatory beta subunit 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.
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kcnab3 (potassium channel, voltage gated subfamily A regulatory beta subunit 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
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