A Genetically Encoded Actuator Selectively Boosts L-type Calcium Channels in Diverse Physiological Settings

Abstract

L-type Ca2+ channels (CaV1.2/1.3) convey influx of calcium ions (Ca2+) that orchestrate a bevy of biological responses including muscle contraction and gene transcription. Deficits in CaV1 function play a vital role in cardiac and neurodevelopmental disorders. Yet conventional pharmacological approaches to upregulate CaV1 are limited, as excessive Ca2+ influx leads to cytotoxicity. Here, we develop a genetically encoded enhancer of CaV1.2/1.3 channels (GeeC) to manipulate Ca2+ entry in distinct physiological settings. Specifically, we functionalized a nanobody that targets the CaV macromolecular complex by attaching a minimal effector domain from a CaV enhancer-leucine rich repeat containing protein 10 (Lrrc10). In cardiomyocytes, GeeC evoked a 3-fold increase in L-type current amplitude. In neurons, GeeC augmented excitation-transcription (E-T) coupling. In all, GeeC represents a powerful strategy to boost CaV1.2/1.3 function in distinct physiological settings and, in so doing, lays the groundwork to illuminate new insights on neuronal and cardiac physiology and disease.

Cardiac L-type calcium channel regulation by Leucine-Rich Repeat-Containing Protein 10

“…Therefore, the interaction between LRRC10 and Ca V 1.2 does not require the β 2 and α 2 δ-1 subunits of LTCC. This finding is recently supported by consistent results from a Förster/fluorescence resonance energy transfer (FRET) assay [ 38 ]. These data, however, do not prove a direct interaction between LRRC10 and Ca V 1.2 but do at least suggest LRRC10 and Ca V 1.2 are associated in a macromolecular complex.…”

“… [ 18 ] Transient expression of rabbit Ca V 1.2 in HEK-293 cells (with auxiliary subunits) FRET, patch clamp LRRC10 interacted with Ca V 1.2 and modulated I Ca,L . [ 38 ] Co-IP, co-immunoprecipitation; FRET, Förster/fluorescence resonance energy transfer; IF, immunofluorescence. …”

“…A recent study [ 38 ] demonstrated that N-terminus-truncated LRRC10 still robustly interacted with Ca V 1.2, but the I Ca,L -potentiating effect was markedly reduced. The N-terminus of LRRC10 alone hardly interacted with Ca V 1.2.…”

“…This fusion protein construct (named “genetically encoded enhancer of Ca 2+ current” or “GeeC”) was capable of increasing I Ca,L in a heterologous expression system (HEK-293 cells), mouse ventricular cardiomyocytes, and mouse cortical and hippocampal neurons. Thus, it was proposed that the N-terminus is the “minimal effector domain” of LRRC10, whereas the rest of the molecule (the LRRs and the C-terminus) serves as the “high-affinity binding domain.” GeeC did not alter plasma membrane expression of LTCC, and single-channel recordings revealed that GeeC upregulated I Ca,L by increasing P O without changing the unitary conductance [ 38 ].…”

“…In sinoatrial nodal cells [ 50 ], Ca V 1.3 can contribute up to ≈50–70% of I Ca,L [ 51 , 52 ]. The N-terminus of LRRC10 in the GeeC construct (see above), being guided to LTCCs by a Ca V β-targeting nanobody, has been reported to augment I Ca,L through Ca V 1.3 in HEK-293 cells [ 38 ]. It will be important to test whether the native full-length LRRC10 is also capable of associating with and modulating Ca V 1.3.…”