Increased phosphorylation of the neuronal L-type Ca(2+) channel Ca(v)1.2 during aging

Abstract

An increase in Ca2+ influx through L-type Ca2+ channels is thought to contribute to neuronal dysfunctions that underlie senile symptoms and Alzheimer's disease. The molecular basis of the age-dependent up-regulation in neuronal L-type Ca2+ channel activity is largely unknown. We show that phosphorylation of the L-type channel Cav1.2 by cAMP-dependent protein kinase is increased >2-fold in the hippocampus of aged rats. The hippocampus is critical for learning and is one of the first brain regions to be affected in Alzheimer's disease. Phosphorylation of Cav1.2 by cAMP-dependent protein kinase strongly enhances its activity. Therefore, increased Cav1.2 phosphorylation may account for a substantial portion of the age-related rise in neuronal Ca2+ influx and its neuropathological consequences.

Fig. 1.

Quantification of α₁1.2 phosphorylation on serine 1928. α₁1.2 was immunoprecipitated with anti-CNC1 from hippocampal Triton X-100 extracts from adult rats for immunoblotting with anti-CH1923-1932P (71). After stripping with SDS and DTT, blots were reprobed with anti-CNC1 (71). (A) Anti-CH1923-1932P recognized α₁1.2 when phosphatase inhibitors were present (lane 2) but not when omitted (lane 1). In vitro phosphorylation of immunoprecipitated α₁1.2 with purified PKA strongly increased the signal (lane 3). (B) Fifty percent (one half of one immunoprecipitate), 100%, and 200% (two immunoprecipitates combined) of immunoprecipitates were used for immunoblotting. Films were exposed for 0.5, 1, and 2 min, and immunosignals were quantified by densitometry (28, 33). Values indicated by the bars reflect the average of three samples. (C) After immunoprecipitation of α₁1.2, one sample but not the other was phosphorylated in vitro with purified PKA under conditions that led to near complete phosphorylation of the PKA site on α₁1.2, as quantified earlier (21). Immunosignals for anti-CH1923-1932P were corrected for differences in relative amounts of long-form α₁1.2 based on the corresponding anti-CNC1 signals. Under basal conditions 16.7 ± 1.8% (SD; n = 3, obtained in three different experiments) of long-form α₁1.2 is phosphorylated on serine 1928 (left bar) with 100% equaling the anti-CH1923-1932P signal obtained after in vitro phosphorylation.

Fig. 2.

Increase in basal phosphorylation of α₁1.2 on serine 1928 in 26-month-old rats. α₁1.2 was immunoprecipitated from hippocampal extracts of 12- and 26-month-old Lobund-Wistar rats. Immunoblots were probed with anti-CH1923-1932P, stripped with SDS and DTT, and reprobed with anti-CNC1 (26). Immunosignals for long- and short-form anti-CH1923-1932P (Top) and anti-CNC1 (Middle) were quantified by densitometry. Signals for anti-CH1923-1932P corrected for differences in relative amounts of α_1C based on the CNC1 signals of the long-form α_1C (Bottom). Values for long- and short-form α₁1.2 were normalized by setting the average values for long- and short-form α₁1.2 in adults as 100%. The average phosphorylation level of serine 1928 is 16.7% in adult rats (see Fig. 3); therefore, we set the average of all values for adult rats from each aging experiment equal to 16.7% and normalized all measured values accordingly. Bars represent average of 16 samples ± SEM. The small decrease in total amount of short-form α₁1.2 in old rats to 89 ± 6% of adult is statistically significant (P < 0.05 by one-way ANOVA and Dunnett's multiple comparison test).

Fig. 3.

MAP2B, NR1, PSD-95, and SAP102 are unchanged in old rats. Hippocampal samples were homogenized in 1% Triton X-100 (A) or 1% deoxycholate (B), and soluble and insoluble proteins were separated by ultracentrifugation. (A) Aliquots of comparable amounts of supernatants (Right) and pellets (Left) were analyzed by immunoblotting with antibodies against MAP2B and NR1 and quantified by densitometry. (B) Aliquots of the supernatants were analyzed either directly by immunoblotting with anti-PSD-95 and anti-SAP102 (Left) or after their coprecipitation with NMDA receptors by using a mixture of anti-NR2A and NR2B antibodies for immunoprecipitation (Right). All values were normalized with respect to the average values for each set of markers from adult rats, which corresponded to 100%. Bars represent average of n samples ± SEM (n is given in each bar).