Identification of the amino terminus of neuronal Ca2+ channel alpha1 subunits alpha1B and alpha1E as an essential determinant of G-protein modulation

Abstract

We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.

Fig. 1.

Sequence of α1E_long used in this study. A, DNA alignment of the 5′ sequences of α1E(rbEII) (L15453), rat α1E_long (AF057029), and mouse α1E (L29346). Shaded areas show translated sequences. The vertical arrow shows the position of the restriction site NotI, which was used to subclone the extended 5′ sequence onto α1E(rbEII). The boxed CGG nucleotides before the ATG start site in the α1E(rbEII) were found to be present in the rbEII clone but are absent from the L15453sequence in the database. This triplet is also present in the published mouse, human, and rabbit α1E sequences. The forward primers used (see Materials and Methods) are shown as horizontal arrows, below (primer 1) or above (primers 2 and3) the corresponding sequence. Note that the extended N-terminal sequence of α1E_long shows a high degree of homology with part of the reported 5′ untranslated sequence of the rbEII cDNA. B, Amino acid alignment for the N termini of rat α1E_long, rabbit α1B (published sequence), and rat α1E(rbEII, published sequence). Conserved residues areshaded. The rat α1E_long N-terminal amino acid sequence was also identical to that of the published mouse α1E sequence (L29346).

Fig. 2.

Properties and G-protein modulation of α1E_long: comparison with α1E(rbEII). Ashows the lack of modulation of α1E(rbEII) in the absence of co-transfected VDCC β subunits. α1E(rbEII) was expressed with α2-δ but without β2a subunits in Xenopus oocytes (together with D2 dopamine receptors). Left panel, Example currents, control (1), plus quinpirole (2), and after a depolarizing prepulse to +100 mV in the presence of quinpirole (3). The voltage protocol is shown above the current traces. Middle panel, Time course of I_Ba amplitude during quinpirole application. Right panel,I–V plot before (○) and during (•) quinpirole application (n = 6). TheI–V data were fitted with a modified Boltzmann equation as described previously (Page et al., 1997).B shows the modulation of α1E_long in the presence of co-transfected VDCC β subunits. α1E_long was expressed with both α2-δ and β2a subunits inXenopus oocytes (together with D2 dopamine receptors). Activation of dopamine D2 receptors by quinpirole (100 nm) in oocytes caused a reversible inhibition ofI_Ba. Left panel, Example currents, control (1), plus quinpirole (2), and after a depolarizing prepulse in the presence of quinpirole (3). Middle panel, Time course of inhibition by quinpirole. Right panel, I–V plot before (○) and during (•) quinpirole application (n = 9). TheI–V data were fitted as described inA. The boxed inset shows the voltage-dependence of the inhibition by quinpirole from theI–V data of α1E_long(solid bars, n = 9). Data for α1B (open bars, n = 8) are plotted for comparison; * p < 0.01 (Student’st test).

Fig. 3.

G-protein modulation of α1E_longexpressed in COS-7 cells. α1E_long was expressed with accessory VDCC α2-δ and β2a subunits in the presence or absence of co-expressed Gβ1γ2. A, Examples of current density–voltage profiles for α1E_long in a control cell in the presence of GDPβS to limit any tonic G-protein modulation (left), and a cell co-expressing Gβ1γ2 (right) (V_t = −40 to −10 mV, in 10 mV steps). B, Voltage-dependence of τ_act for α1E_long with co-expressed Gβ1γ2 (•, n = 10), α1E_long in the presence of GDPβS (□, n = 7), and α1E(rbEII) with co-expressed Gβ1γ2 (○, n = 5), * p < 0.01 compared with respective control. C, Example of facilitation of α1E_longI_Ba in the presence of co-expressed Gβ1γ2 by a depolarizing prepulse to +120 mV, 10 msec before and immediately after equivalent test pulses P1 and P2, to test potentials (V_t) between −40 and −10 mV in 10 mV intervals. The voltage protocol is shown above the current traces. Facilitation was then determined as the P2/P1 ratio of the current amplitudes in P1 and P2 (Table 1).

Fig. 4.

G-protein modulation of an α1E construct containing the N terminus of α1B. A, The α1 subunit construct in which the α1B_1–55 sequence was added to α1E(rbEII) to form α1bEEEE was expressed with accessory VDCC α2-δ and β2a subunits in Xenopus oocytes (together with D2 receptors) or in COS-7 cells (together with Gβ1γ2 subunits). B, α1bEEEE currents expressed in oocytes.Left panel, Example currents, control (1), plus quinpirole (2), and after a depolarizing prepulse in the presence of quinpirole (3). The voltage protocol is the same as shown in Figure 2A. Middle panel, Time course of inhibition by quinpirole. Right panel,I–V plot before (○) and during (•) quinpirole application (n = 9). TheI–V data were fitted according to the legend to Figure 2. The boxed inset shows the voltage-dependence of the inhibition by quinpirole from theI–V data (open bars,n = 9). Data for α1E_long(solid bars, n = 9) are plotted for comparison; * p < 0.05 (Student’st test). C, α1bEEEE currents expressed in COS-7 cells. Left panel, Example current density–voltage profiles for control α1bEEEEI_Ba in the presence of 2 mmGDPβS. Middle panel, α1bEEEEI_Ba in the presence of Gβ1γ2 (V_t = −40 to −10 mV in 10 mV steps).Right panel, Voltage-dependence of τ_actfor α1bEEEE in the presence (•, n = 5) or absence (○, n = 3) of co-expressed Gβ1γ2; *p < 0.01 compared with respective control.

Fig. 5.

Lack of G-protein modulation of an N-terminally truncated α1B construct. A, The α1 construct in which the α1B_1–55 sequence was deleted from α1B to form α1BΔN_1–55 was expressed with accessory VDCC α2-δ and β2a subunits in Xenopus oocytes (together with D2 receptors) or in COS-7 cells (together with Gβ1γ2 subunits). B, α1BΔN_1–55 currents expressed in oocytes. Left panel, Example currents, control (1), plus quinpirole (2), and after a depolarizing prepulse in the presence of quinpirole (3). The voltage protocol is the same as shown in Figure 2A. Middle panel, Time course of I_Ba amplitude during quinpirole application. Right panel,I–V plot before (○) and during (•) quinpirole application (n = 7). TheI–V data were fitted according to the legend to Figure 2. C, α1BΔN_1–55currents expressed in COS-7 cells. Left panel, Example current density–voltage profiles in the absence or presence of Gβ1γ2 (V_t = −40 to −10 mV in 10 mV steps). Right panel, Voltage-dependence of τ_act in the presence (•, n = 10) or absence (○, n = 7) of co-expressed Gβ1γ2.

Fig. 6.

Reinhibition kinetics of α1E_long and α1B. Prepulses of 50 msec duration to +100 mV were applied, and the time between prepulse and test pulse to 0 mV (interpulse interval Δt at −100 mV) was increased, in 10 msec steps, up to 220 msec. There was no difference between the τ_reinhibition for α1E_long (○,n = 9) and α1B (•, n = 9)I_Ba.