Abstract
L-type calcium channels (LTCCs) are highly expressed in the heart and brain and are critical for cardiac and neuronal functions. LTCC-blocking drugs have a long and successful record in the clinic for treating cardiovascular disorders. In contrast, establishment of their efficacy for indications of the central nervous system remains challenging given the tendency of existing LTCC drugs being functionally and mechanistically more selective for peripheral tissues. LTCCs in vivo are large macromolecular complexes consisting of a pore-forming subunit and other modulatory proteins, some of which may be neuro-specific and potentially harbor mechanisms for neuronal selectivity. To exploit the possibility of identifying mechanistically novel and/or neuro-selective blockers, we developed two phenotypic assays—a calcium flux-based primary screening assay and a patch clamp secondary assay, using rat primary cortical cultures. We screened a library comprised of 1278 known bioactive agents and successfully identified a majority of the potent LTCC-blocking drugs in the library. Significantly, we identified a previously unrecognized LTCC blocker with a novel mechanism, which was corroborated by patch clamp and binding studies. As such, these phenotypic assays are robust and represent an important step towards identifying mechanistically novel and neuro-selective LTCC blockers.
Highlights
L-type calcium channels (LTCCs) are highly expressed in the heart and brain and are critical for cardiac and neuronal functions
voltage-gated calcium channels (VGCCs) in vivo are large macromolecular signaling complexes consisting of a central pore-forming α1 subunit surrounded by multiple auxiliary subunits (e.g., β and α2δ) and other interacting/modulatory proteins
In contrast to 100 mM K+, the Bay K8644 (1.1 μM)-induced response was completely blocked by 3 μM nimodipine (Fig. 1a), indicating that it was LTCC mediated
Summary
L-type calcium channels (LTCCs) are highly expressed in the heart and brain and are critical for cardiac and neuronal functions. LTCCs in vivo are large macromolecular complexes consisting of a pore-forming subunit and other modulatory proteins, some of which may be neuro-specific and potentially harbor mechanisms for neuronal selectivity. We identified a previously unrecognized LTCC blocker with a novel mechanism, which was corroborated by patch clamp and binding studies. Establishment of the clinical efficacy of LTCC-blocking drugs remains challenging for disorders of the central nervous system (CNS), such as bipolar disorder[7]. All known LTCC drugs bind to the pore region of α1 subunits, functional potency in vivo appears to vary substantially in the brain and cardiovasculature. It is conceivable that LTCCs in neurons may confer novel and/ or neuro-selective pharmacological properties through neuro-favoring/specific interactions
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