SUMMARY Regulation of neuronal excitability and cardiac excitation-contraction coupling requires proper localization of L-type Ca2+ channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming CaV1.2 subunit, to foster its surface expression. Disruption of α-actinin function by dominant negative or shRNA constructs reduces CaV1.2 surface localization in HEK293 and neuronal cultures, and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca2+ influx through L-type channels but not through NMDAR, thereby triggering loss of CaV1.2 from spines. Coexpression of a Ca2+-binding deficient calmodulin mutant does not affect basal CaV1.2 surface expression, but inhibits its internalization upon Ca2+ influx. We conclude that α-actinin stabilizes CaV1.2 at the plasma membrane, and that its displacement by Ca2+-calmodulin induces Ca2+-induced endocytosis of CaV1.2, thus providing an important negative feedback mechanism for Ca2+ influx.

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Cav1 (L-type) channels and calmodulin-dependent protein kinase II (CaMKII) are key regulators of Ca2+signaling in neurons. CaMKII directly potentiates the activity of Cav1.2 and Cav1.3 channels, but the underlying molecular mechanisms are incompletely understood. Here, we report that the CaMKII-associated protein densin is required for Ca2+-dependent facilitation of Cav1.3 channels. While neither CaMKII nor densin independently affects Cav1.3 properties in transfected HEK293T cells, the two together augment Cav1.3 Ca2+currents during repetitive, but not sustained, depolarizing stimuli. Facilitation requires Ca2+, CaMKII activation, and its association with densin, as well as densin binding to the Cav1.3 α1subunit C-terminal domain. Cav1.3 channels and densin are targeted to dendritic spines in neurons and form a complex with CaMKII in the brain. Our results demonstrate a novel mechanism for Ca2+-dependent facilitation that may intensify postsynaptic Ca2+signals during high-frequency stimulation.

Background Voltage-gated (Cav) Ca2+ channels are multi-subunit complexes that play diverse roles in a wide variety of tissues. A fundamental mechanism controlling Cav channel function involves the Ca2+ ions that permeate the channel pore. Ca2+ influx through Cav channels mediates feedback regulation to the channel that is both negative (Ca2+-dependent inactivation, CDI) and positive (Ca2+-dependent facilitation, CDF). Scope of Review This review highlights general mechanisms of CDI and CDF with an emphasis on how these processes have been studied electrophysiologically in native and heterologous expression systems. Major Conclusions Electrophysiological analyses have led to detailed insights into the mechanisms and prevalence of CDI and CDF as Cav channel regulatory mechanisms. All Cav channel family members undergo some form of Ca2+-dependent feedback that relies on CaM or a related Ca2+ binding protein. Tremendous progress has been made in characterizing the role of CaM in CDI and CDF. Yet, what contributes to the heterogeneity of CDI/CDF in various cell-types and how Ca2+-dependent regulation of Cav channels controls Ca2+ signaling remain largely unexplored. General Significance Ca2+ influx through Cav channels regulates diverse physiological events including excitation-contraction coupling in muscle, neurotransmitter and hormone release, and Ca2+-dependent gene transcription. Therefore, the mechanisms that regulate channels, such as CDI and CDF, can have a large impact on the signaling potential of excitable cells in various physiological contexts.

Key points• In the sinoatrial node (SAN), Ca v 1 voltage-gated Ca 2+ channels mediate L-type currents that are essential for normal cardiac pacemaking.• Both Ca v 1.2 and Ca v 1.3 Ca 2+ channels are expressed in the SAN but how their distinct properties affect cardiac pacemaking is unknown.• Here, we show that unlike Ca v 1.2, Ca v 1.3 undergoes voltage-dependent facilitation and colocalizes with ryanodine receptors in sarcomeric structures.• By mathematical modelling, these properties of Ca v 1.3 can improve recovery of pacemaking after pauses and stabilize SAN pacemaking during excessively slow heart rates.• We conclude that voltage-dependent facilitation and colocalization with ryanodine receptors distinguish Ca v 1.3 from Ca v 1.2 channels in the SAN and contribute to the major impact of Ca v 1.3 on pacemaking.Abstract Dysregulation of L-type Ca 2+ currents in sinoatrial nodal (SAN) cells causes cardiac arrhythmia. Both Ca v 1.2 and Ca v 1.3 channels mediate sinoatrial L-type currents. Whether these channels exhibit differences in modulation and localization, which could affect their contribution to pacemaking, is unknown. In this study, we characterized voltage-dependent facilitation (VDF) and subcellular localization of Ca v 1.2 and Ca v 1.3 channels in mouse SAN cells and determined how these properties of Ca v 1.3 affect sinoatrial pacemaking in a mathematical model. Whole cell Ba 2+ currents were recorded from SAN cells from mice carrying a point mutation that renders Ca v 1.2 channels relatively insensitive to dihydropyridine antagonists. The Ca v 1.2-mediated current was isolated in the presence of nimodipine (1 μM), which was subtracted from the total current to yield the Ca v 1.3 component. With strong depolarizations (+80 mV), Ca v 1.2 underwent significantly stronger inactivation than Ca v 1.3. VDF of Ca v 1.3 was evident during recovery from inactivation at a time when Ca v 1.2 remained inactivated. By immunofluorescence, Ca v 1.3 colocalized with ryanodine receptors in sarcomeric structures while Ca v 1.2 was largely restricted to the delimiting plasma membrane. Ca v 1.3 VDF enhanced recovery of pacemaker activity after pauses and positively regulated pacemaking during slow heart rate in a numerical model of mouse SAN automaticity, including preferential coupling of Ca v 1.3 to ryanodine receptor-mediated Ca 2+ release. We conclude that strong VDF and colocalization with