A Xenopus oocyte β 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 ␤ 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 ␤ antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca 2؉ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian ␣ 1 cRNAs (␣ 1C and ␣ 1E ); (ii) coinjection of a Xenopus ␤ antisense oligonucleotide and excess rat ␤ cRNA rescued expression of ␣ 1 Ca 2؉ channel currents; and (iii) coinjection of mammalian ␣ 1 cRNA with cRNA encoding either of the two Xenopus ␤ subunits facilitated both activation and inactivation of Ca 2؉ channel currents by voltage, as happens with most mammalian ␤ subunits. The Xenopus ␤ subunit cDNAs (␤3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 ␤ subunit. We propose that ''␣ 1 alone'' channels are in fact tightly associated ␣ 1 ␤3xo channels, and that effects of exogenous ␤ subunits are due to formation of higher-order [␣ 1 ␤]␤ n complexes with an unknown contribution of ␤3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition ␣ 1 ␤, are [␣ 1 ␤]␤ n complexes that associate with ␣ 2 ␦ and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of ␣ 1E have a ␤ subunit binding domain. This ␤ binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel ␣ 1 subunits.Xenopus oocytes translate exogenously injected mRNAs and cRNAs with relatively high efficiency. This has made them systems of choice for the functional expression and characterization of many cloned molecules, such as neuronal ligandgated ion channels, G protein-coupled receptors, and many voltage-gated ion channels, including voltage-dependent Ca 2ϩ channels. Voltage-dependent Ca 2ϩ channels are formed of an ␣ 1 pore-forming and voltage-sensing subunit and ␤ and ␣ 2 ␦ regulatory subunits. Functional expression in Xenopus oocytes has not only been used to define structure-function relations of voltage-gated calcium channels by assessing the effects of specific mutations of the ␣ 1 channel protein, but also to define identity and roles of the regulatory subunits in promoting ␣ 1 expression or modifying the properties of the expressed ␣ 1 subunit. Several nonallelic genes encoding ␣ 1 subunits, termed ␣ 1S and ␣ 1A -␣ 1E , have been identified by molecular cloning (1-4). Of these, all except ␣ 1S have been functionally expressed in the Xenopus oocyte. However, while certain variants of ␣ 1C and ␣ 1E can be expressed without coinjection of other subunit cRNAs (e.g., refs. 5 and 6), others, particularly ␣ 1A , yield only minimal currents in the absence of additional subunits, notably a ␤ subunit (7,8). The reasons for these differences are not understood.The interpreta...

Bimolecular fluorescence complementation and targeted biotinylation provide insight into the topology of the skeletal muscle Ca2+channel β1a subunit

“…95%) may have contributed to this expression. 36 Figure 8 presents a tentative model of the β1a subunit and its arrangement in DHPR tetrads. Because the SH3 and GK domains are highly conserved among all Ca V β subunits, [15][16][17] it is assumed that their structure in β1a is like that determined crystallographically for β2a.…”

Differential Interactions of the C terminus and the Cytoplasmic I-II Loop of Neuronal Ca2+ Channels with G-protein α and βγ Subunits

“…Moreover, currents through the mutant channel, B3L⌬2, were not detectable in the absence of Ca 2ϩ channel ␤ subunit expression (n ϭ 5). Because B3L⌬2 was devoid of the segment corresponding to the major binding site for the ␤ subunit (31), this indicates that there may be another interaction site on the ␣ 1B channel for ␤ subunits (32).…”

Alternative Splicing of the β4Subunit Has α1Subunit Subtype-Specific Effects on Ca2+Channel Gating

“…Critical to the interpretation of our expression data is the fact that some populations of Xenopus oocytes have been shown to express low levels of an endogenous ␤ 3 -like subunit that is capable of binding to and altering the gating properties of injected ␣ 1 subunits (Tareilus et al, 1997). To test for this possibility in our oocytes, we conducted experiments in which we measured the time required for ␣ 1A /␣ 2 ␦, ␣ 1A /␣ 2 ␦ ϩ ␤ 4a , and ␣ 1A /␣ 2 ␦ ϩ ␤ 4b complexes to reach levels of expression that we thought suitable for electrophysiological recording (1 A of peak current).…”