A Xenopus oocyte beta subunit: evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit

Abstract

Two closely related beta subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus beta antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca2+ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian alpha1 cRNAs (alpha(1C) and alpha(1E)); (ii) coinjection of a Xenopus beta antisense oligonucleotide and excess rat beta cRNA rescued expression of alpha1 Ca2+ channel currents; and (iii) coinjection of mammalian alpha1 cRNA with cRNA encoding either of the two Xenopus beta subunits facilitated both activation and inactivation of Ca2+ channel currents by voltage, as happens with most mammalian beta subunits. The Xenopus beta subunit cDNAs (beta3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 beta subunit. We propose that "alpha1 alone" channels are in fact tightly associated alpha1beta3xo channels, and that effects of exogenous beta subunits are due to formation of higher-order [alpha1beta]beta(n) complexes with an unknown contribution of beta3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition alpha1beta, are [alpha1beta]beta(n) complexes that associate with alpha2delta and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of alpha(1E) have a beta subunit binding domain. This beta binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel alpha1 subunits.

Figure 1

Amino acid sequence of Xenopus β subunits as deduced from the xo28 and xo32 cDNAs and comparison to sequences of mammalian β1b, β2a, β2b, β3, and β4 (A) and phylogenetic tree of calcium channel β subunits (B). (A) xoβ₃-28 is the reference. –, Same amino acid as in xoβ₃-28; –, gap; @, stop; rt, rat; rb, rabbit; hum, human; and xo, X. laevis oocyte. β2a differs from β2b only at the N terminus. β1, β1b splice variant. The phylogram shown in B was calculated by the neighbor-joining technique of Kimura, using version 8.0 of the gcg Sequence Analysis Software Package. Numbers in parenthesis next to names of β subunits are the GenBank accession numbers: for xo28, it is U33217U33217, and for xo32, it is U33218U33218.

Figure 2

Effect of β3xo (xo28) on α_1E. (A and B) Time courses of activation of α_1E in oocytes injected with α_1E cRNA alone or cRNAs encoding both α_1E and β3xo (clone xo28). (C and D) Effect of β3xo on voltage-dependent activation and inactivation of α_1E. G–V curves were obtained from peak tail currents measured by stepping to −50 mV after depolarizing pulses of 25-msec duration from −88 to 116 mV in 4-mV increments. The data were sampled at 10 kHz and filtered at 2 kHz. The data points were fitted by the sum of two Boltzmann distributions. For α_1E, the first component had a V_1/2 = 2.7 mV, an effective valence (zδ) = 2.9 e, and a relative amplitude of 51%; the second had a V_1/2 = 41.8 mV and a zδ = 1.4 e. For α_1E + β3xo, the first component had a V_1/2 = 2.7 mV, a zδ = 3.4 e and a relative amplitude of 75%; the second had a V_1/2 = 51.8 mV and a zδ = 1.3 e. Steady-state inactivation curves were derived from peak currents elicited by a pulse to +20 mV following a conditioning pulse of 10 sec to potentials from −120 to 27 mV in 7 mV increments and a brief (4-msec) pulse to −90 mV. The data were sampled at 500 Hz and filtered at 100 Hz. Sweeps were separated by 20 sec to allow a full recovery from inactivation. Data points were fitted by a Boltzmann distribution. The effective valences were 2.7 ± 0.1 e for α_1E and 3.4 ± 0.1 e for α_1E + β3xo; the half-inactivation potentials were −32.2 ± 3.4 mV (n = 5) for α_1E and −53.5 ± 1.1 mV (n = 4) for α_1E + β3xo.

Figure 3

Inhibition by anti-β3xo oligonucleotide B11 of endogenous Ca²⁺ channel currents in oocytes of one frog presenting such currents. Expression of Ca²⁺ channel currents in Xenopus oocytes was determined on the day after isolation and collagenase treatment of oocytes. Oocytes from one frog expressing 5–10 nA at +30 mV with 74 mM Ba²⁺ in the external solution were injected with 50 nl of 100 μM B11 (antisense) or B10 (sense) oligonucleotide. The injected oocytes were then tested 3, 5, and 7 days later for Ca²⁺ channel activity. (A) Representative I_Ba recorded from one uninjected oocyte. (B) I–V relations obtained 7 days after injection of sense or antisense oligonucleotides. The external solution was 80 mM Ba²⁺/10 mM Hepes, titrated to pH 7.0 with methanesulfonic acid (CH₃SO₃H).

Figure 4

Inhibition by anti-β3xo oligonucleotide B24 of expression of α_1E currents and rescue of α_1E currents by coinjection of cRNA encoding the rat β1b subunit. Oocytes were injected with 50 nl of a solution containing 100 μg/ml of α_1E cRNA alone or in combination with 100 μg/ml β1b cRNA and the indicated concentrations of B24 oligonucleotide. Ca²⁺ channel currents were recorded 6 days after injection. The results presented on this figure were obtained with oocytes obtained from a single frog. Similar results but varying in the concentration of B24 needed to affect α_1E currents, were obtained in two other experiments. (A) Peak inward α_1E currents recorded from oocytes after a 250-msec test pulse from a holding potential of −90 mV. Injection of cRNAs and B24 oligonucleotide are indicated above the bars, which are means ± SEM of 4–5 oocytes. (B) Averaged G–V curves (mean ± SEM) of α_1E currents in oocytes coinjected or not with B24 and B24 plus β1b cRNA. The composition of B24 is given in Table 1.

Figure 5

Identification of two sites on α_1E that interact with β subunits. (A) Ideogram of a Ca²⁺ channel α₁ subunit with linear N and C termini, four homologous repeats (filled boxes), and connecting loops. (B) 12% SDS/PAGE analysis of GST fusion proteins and bound calcium channel β2a or G protein α_s subunits synthesized by reticulocyte lysates in the presence of [³⁵S]methionine. BI, Coomassie blue stain; BII, autoradiogram of the gel shown in BI. (C) Experimental design of the test for protein–protein interaction. The figure shows binding of β_2a to the L1 and the CC regions of α_1E. L1, α_1E[356–451]; and CC, α_1E[2036–2312]. Note that both α_1E L1 and α_1E CC bound [³⁵S]CCβ_2a but not an unrelated protein, [³⁵S]G_sα.