What is the clinical evidence from this study?

Study design: Other. Intervention: CH2II helix deletion (ΔCH2 mutant) vs. Wild-type Cav2.3 channel. Primary outcome: Half-maximal steady-state inactivation (V1/2).

November 30, 2022Open Access

Structures of the R-type human Cav2.3 channel reveal conformational crosstalk of the intracellular segments

XYXia YaoWuhan University YWYan WangSt. John's University ZWZhifei WangSt. John's University

Key Result

Deletion of the CH2II helix in the human Cav2.3 channel shifted the half-maximal steady-state inactivation from -70.9 mV to -57.3 mV, demonstrating that CH2II stabilizes the inactivated conformation.

Structured PICO

Population

Human Cav2.3 channel (recombinantly expressed in HEK293F cells)

Intervention

Deletion of the CH2II helix (ΔCH2 mutant)

Comparator

Wild-type human Cav2.3 channel

Outcome

Cryo-EM structure and electrophysiological properties (activation, steady-state inactivation, recovery from inactivation)surrogate

The cryo-EM structure of human Cav2.3 reveals that the CH2II helix stabilizes the inactivated state of the channel, providing a structural basis for potential drug design.

Main Result

Absolute Event Rate: -57.3% vs -70.9%

Limitations

The β3 subunit and intracellular segments were barely discernible in the high-resolution map of the ΔCH2 mutant.
No discernible density for the SNX-482 peptide toxin was observed despite its addition during purification.
Cannot definitively confirm if the density seen in low pass-filtered maps of the ΔCH2 mutant belongs to the β3 subunit.

Abstract

Abstract The R-type voltage-gated Ca 2+ (Ca v ) channels Ca v 2.3, widely expressed in neuronal and neuroendocrine cells, represent potential drug targets for pain, seizures, epilepsy, and Parkinson’s disease. Despite their physiological importance, there have lacked selective small-molecule inhibitors targeting these channels. High-resolution structures may aid rational drug design. Here, we report the cryo-EM structure of human Ca v 2.3 in complex with α2δ−1 and β3 subunits at an overall resolution of 3.1 Å. The structure is nearly identical to that of Ca v 2.2, with VSD II in the down state and the other three VSDs up. A phosphatidylinositol 4,5-bisphosphate (PIP2) molecule binds to the interface of VSD II and the tightly closed pore domain. We also determined the cryo-EM structure of a Ca v 2.3 mutant in which a Ca v 2-unique cytosolic helix in repeat II (designated the CH2 II helix) is deleted. This mutant, named ΔCH2, still reserves a down VSD II , but PIP2 is invisible and the juxtamembrane region on the cytosolic side is barely discernible. Our structural and electrophysiological characterizations of the wild type and ΔCH2 Ca v 2.3 show that the CH2 II helix stabilizes the inactivated conformation of the channel by tightening the cytosolic juxtamembrane segments, while CH2 II helix is not necessary for locking the down state of VSD II .

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