Dataset Information

Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels.

ABSTRACT: Genetically encoded inhibitors for voltage-dependent Ca²⁺ (Ca_V) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca²⁺ (Ca_V1/Ca_V2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary Ca_V?-subunits. To investigate latent Ca_V?-independent components of inhibition, we coexpressed each RGK individually with Ca_V1 (Ca_V1.2/Ca_V1.3) or Ca_V2 (Ca_V2.1/Ca_V2.2) channels reconstituted in HEK293 cells with either wild-type (WT) ?_2a or a mutant version (?_2a,TM) that does not bind RGKs. All four RGKs strongly inhibited Ca_V1/Ca_V2 channels reconstituted with WT ?_2a By contrast, when channels were reconstituted with ?_2a,TM, Rem inhibited only Ca_V1.2, Rad selectively inhibited Ca_V1.2 and Ca_V2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT Ca_V?, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted Ca_V1.2 while Rad[R208A/L235A] inhibited Ca_V1.2/Ca_V2.2 but not Ca_V1.3/Ca_V2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous Ca_V1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for Ca_V1.2 and Ca_V1.2/Ca_V2.2 channels. Thus, we have exploited latent ?-binding-independent Rem and Rad inhibition of specific Ca_V1/Ca_V2 channels to develop selective GECCIs with properties unmatched by current small-molecule Ca_V channel blockers.

SUBMITTER: Puckerin AA

PROVIDER: S-EPMC6255209 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels.

Puckerin Akil A AA Chang Donald D DD Shuja Zunaira Z Choudhury Papiya P Scholz Joachim J Colecraft Henry M HM

Proceedings of the National Academy of Sciences of the United States of America 20181105 47

Genetically encoded inhibitors for voltage-dependent Ca2+ (CaV) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca2+ (CaV1/CaV2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary CaV</su ...[more]

PMID: 30397133

Similar Datasets

Project description:How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4-12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel.

Dataset Information

Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels.

Publications

Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets