Molecular diversity of the calcium channel alpha2delta subunit - PubMed (original) (raw)

Molecular diversity of the calcium channel alpha2delta subunit

N Klugbauer et al. J Neurosci. 1999.

Abstract

Sequence database searches with the alpha2delta subunit as probe led to the identification of two new genes encoding proteins with the essential properties of this calcium channel subunit. Primary structure comparisons revealed that the novel alpha2delta-2 and alpha2delta-3 subunits share 55.6 and 30.3% identity with the alpha2delta-1 subunit, respectively. The number of putative glycosylation sites and cysteine residues, hydropathicity profiles, and electrophysiological character of the alpha2delta-3 subunit indicates that these proteins are functional calcium channel subunits. Coexpression of alpha2delta-3 with alpha1C and cardiac beta2a or alpha1E and beta3 subunits shifted the voltage dependence of channel activation and inactivation in a hyperpolarizing direction and accelerated the kinetics of current inactivation. The kinetics of current activation were altered only when alpha2delta-1 or alpha2delta-3 was expressed with alpha1C. The effects of alpha2delta-3 on alpha1C but not alpha1E are indistinguishable from the effects of alpha2delta-1. Using Northern blot analysis, it was shown that alpha2delta-3 is expressed exclusively in brain, whereas alpha2delta-2 is found in several tissues. In situ hybridization of mouse brain sections showed mRNA expression of alpha2delta-1 and alpha2delta-3 in the hippocampus, cerebellum, and cortex, with alpha2delta-1 strongly detected in the olfactory bulb and alpha2delta-3 in the caudate putamen.

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Figures

Fig. 1.

Fig. 1.

a, Amino acid alignment of the α2δ-1 (1), α2δ-2 (2), and α2δ-3 (3) subunits. The N-terminal region differing between the α2δ-2 subunit isoform I and II is_underlined_. Regions that are identical in all sequences are boxed, and conserved cysteine residues are additionally highlighted. The presumptive signal peptides for classes 1 and 3 are shown in italics. Potential N_-glycosylation sites are printed in_bold. The arrow indicates the cleavage site between the α2 and δ proteins of the α2δ-1 subunit. These sequence data are available from the EMBL database under accession numbers M21948 for the α2δ-1 subunit, AF042792 for the α2δ-2(I) subunit, and AF042793 for the α2δ-2(II) subunit isoforms, respectively, and AJ010949for the α2δ-3 subunit. b, Hydrophobicity profile of the α2δ subunits computed according to Kyte and Doolittle (1982). The curve is the average of a residue-specific hydrophobicity index over a window of nine residues.

Fig. 2.

Fig. 2.

Northern blot analysis of the α2δ-2 and α2δ-3 subunits. For each lane, ∼2 μg of poly(A) RNA was run on a denaturing formaldehyde-containing agarose gel, transferred to a nylon membrane, and fixed by UV irradiation. a, Human multiple tissue blot using a specific probe for the α2δ-2 subunit.b, Mouse multiple tissue blot for the α2δ-3 subunit. Arrows indicate predominant species of mRNA with sizes of 5.2 (α2δ-2) and 4.3 (α2δ-3) kb.

Fig. 3.

Fig. 3.

Autoradiographs of α2δ-1 and α2δ-3 riboprobe hybridization to horizontal mouse brain sections. Central (a, c) and more basal (b, d) sections of the brain are shown. Expression of α2δ-1 is seen in the (a) hippocampus (H), cerebral cortex (Co), cerebellum (Ce), and (b) olfactory bulb (Ob). α2δ-3 mRNA was detected in the caudate putamen (CPu), hippocampus (H), entorhinal complex (E), cortex (Co), and thalamic nuclei (T) (c, d).

Fig. 4.

Fig. 4.

The α2δ subunit affects current through the α1C-type calcium channel. The α2δ-3 subunit was coexpressed with α1C and β2a. For comparison, the current through cells coexpressing α2δ-1 subunit was coanalyzed.a shows the voltage dependence of current activation measured as the amplitude of current activated by a 40-msec-long depolarizing pulse from a holding potential of −80 mV to voltages marked on the ordinate and normalized to the maximal amplitude. Each voltage dependence was fitted to the Boltzmann equation. Results of these fits are summarized in Table 1. The inset in the_top left_ of a shows voltage dependence of the kinetics of current activation. The ascending phase of the current time course was fitted to single exponential. The resulting time constants were averaged and plotted against corresponding membrane potentials. In both graphs, ○ represents the α1Cβ2a channel, • the α1Cβ2aα2δ-1 channel, and ▪ the α1Cβ2aα2δ-3 channel. The_inset_ in the right of _a_illustrates a typical family of currents measured during a series of depolarizing pulses from the holding potential of −80 mV to membrane potentials ranging from −20 to +70 mV with a step of +10 mV. ○, α1Cβ2a channel. The cell capacity was 83 pF, and the resulting maximal current density was approximately −13 pA/pF; ▪, α1Cβ2aα2δ-3. The cell capacity was 31 pF, and the corresponding maximal current density was approximately −41 pA/pF. b shows averaged steady-state inactivation curves measured from a holding potential of −80 mV. Current was inactivated by a 5-sec-long prepulse to the potentials marked on the ordinate. This was followed by a 5-msec-long return to the holding potential and a 40-msec-long test pulse to the maximum of the current–voltage relationship. Solid lines are fitted to the Boltzmann equation. The inset shows the time course of the current during a 5-sec-long depolarizing pulse to +20 mV with zero level indicated by a horizontal line. Currents shown are averaged time courses from 9 to 12 experiments scaled to the same amplitude. Individual measurements were fitted to the sum of two exponentials. Results of all fitting procedures are summarized in Table1. Symbols are as in a.

Fig. 5.

Fig. 5.

The α2δ subunit affects current through the α1E-type calcium channel. Unless otherwise indicated, the voltage protocols used were the same as those described in the legend to Figure 4. ○ represents the α1Eβ3 channel; • the α1Eβ3α2δ-1 channel, and ▪ the α1Eβ3α2δ-3 channel. Boltzmann fits of voltage dependencies of current activation are summarized in Table 1. The inset in the_right_ of a shows a typical family of currents measured during a series of depolarizing pulses from a holding potential of −100 mV to membrane potentials ranging from −30 mV to +60 mV with step of +10 mV. ○, α1Eβ3 channel, with a cell capacity of 19 pF and a maximal current density of approximately −34 pA/pF; ▪, α1Eβ3α2δ-3 channel, with a capacity of 43 pF and a maximal current density of approximately −74 pA/pF. b shows the steady-state inactivation curve measured from a holding potential of −100 mV using a 5-sec- long conditioning pulse to membrane potentials marked on the ordinate.Solid lines represent Boltzmann fits. The_insert_ illustrates the inactivation of_I_Ba during a 300-msec-long depolarizing pulse from a holding potential of −100 mV to +20 mV scaled to the same amplitude. Eight to 10 measurements were averaged for each channel type. The α1Eβ3α2δ-1 and α1Eβ3α2δ-3 current traces are indistinguishable from each other. Individual time courses of current inactivation were fitted to a single exponential with a small proportion of noninactivating current. Results of all fits are summarized in Table 1. Symbols are as in_a_.

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