Widespread release of norepinephrine (NE) throughout the forebrain fosters learning and memory via adrenergic receptor (AR) signaling, but the molecular mechanisms are largely unknown. The β₂ AR and its downstream effectors, the trimeric stimulatory G_s-protein, adenylyl cyclase (AC), and the cAMP-dependent protein kinase A (PKA), form a unique signaling complex with the L-type Ca²⁺ channel (LTCC) Ca_V1.2. Phosphorylation of Ca_V1.2 by PKA on Ser¹⁹²⁸ is required for the upregulation of Ca²⁺ influx on β₂ AR stimulation and long-term potentiation induced by prolonged theta-tetanus (PTT-LTP) but not LTP induced by two 1-s-long 100-Hz tetani. However, the function of Ser¹⁹²⁸ phosphorylation in vivo is unknown. Here, we show that S1928A knock-in (KI) mice of both sexes, which lack PTT-LTP, express deficiencies during initial consolidation of spatial memory. Especially striking is the effect of this mutation on cognitive flexibility as tested by reversal learning. Mechanistically, long-term depression (LTD) has been implicated in reversal learning. It is abrogated in male and female S1928A knock-in mice and by β₂ AR antagonists and peptides that displace β₂ AR from Ca_V1.2. This work identifies Ca_V1.2 as a critical molecular locus that regulates synaptic plasticity, spatial memory and its reversal, and LTD.SIGNIFICANCE STATEMENT We show that phosphorylation of the Ca²⁺ channel Ca_V1.2 on Ser¹⁹²⁸ is important for consolidation of spatial memory and especially its reversal, and long-term depression (LTD). Identification of Ser¹⁹²⁸ as critical for LTD and reversal learning supports the model that LTD underlies flexibility of reference memory.

Rationale

β₁ARs (β₁-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β₁AR in cardiac contractility remains to be elucidated.

Objective

Test localization and function of intracellular β₁AR on cardiac contractility.

Methods and results

Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β₁ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca²⁺-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility.

Conclusions

Functional β₁ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β₁ARs requires catecholamine transport via OCT3.

Cocaine-associated contextual cues can trigger relapse behavior by recruiting the hippocampus. Extinction of cocaine-associated contextual memories can reduce cocaine-seeking behavior, however the molecular mechanisms within the hippocampus that underlie contextual extinction behavior and subsequent reinstatement remain poorly understood. Here, we extend our previous findings for a role of Ca_v1.2 L-type Ca²⁺ channels in dopamine 1 receptor (D1R)-expressing cells in extinction of cocaine conditioned place preference (CPP) in adult male mice. We report that attenuated cocaine CPP extinction in mice lacking Ca_v1.2 channels in D1R-expressing cells (D1^cre, Ca_v1.2^fl/fl) can be rescued through chemogenetic activation of D1R-expressing cells within the dorsal dentate gyrus (dDG), but not the dorsal CA1 (dCA1). This is supported by the finding that Ca_v1.2 channels are required in excitatory cells of the dDG, but not in the dCA1, for cocaine CPP extinction. Examination of the role of S1928 phosphorylation of Ca_v1.2, a protein kinase A (PKA) site using S1928A Ca_v1.2 phosphomutant mice revealed no extinction deficit, likely due to homeostatic scaling up of extinction-dependent S845 GluA1 phosphorylation in the dDG. However, phosphomutant mice failed to show cocaine-primed reinstatement which can be reversed by chemogenetic manipulation of excitatory cells in the dDG during extinction training. These findings outline an essential role for the interaction between D1R, Ca_v1.2, and GluA1 signaling in the dDG for extinction of cocaine-associated contextual memories.