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Selective fluorophore-assisted light inactivation of voltage-gated calcium channels.

ABSTRACT: Fluorophore-assisted light inactivation (FALI) is an investigative tool to inactivate fluorescently labeled proteins by a mechanism of in situ photodestruction. We found that Ca(v)1.2 (L-type) and Ca(v)3.1 (T-type) calcium channels, labeled by genetic fusion with GFP derivatives, show differential sensitivity to FALI. Specifically, FALI silences Ca(v)1.2 calcium channels containing EYFP-labeled ?(1C)subunits but does not affect the EYFP-?(1G) Ca(v)3.1 calcium channels or Ca(v)1.2 channels containing EYFP-labeled ? subunits. Our findings limit the applicability of acceptor photobleaching for the measurements of FRET but open an opportunity to combine the fluorescent imaging of the live cell expressing labeled calcium channels with selective functional inactivation of their specific subsets.

SUBMITTER: Kobrinsky E

PROVIDER: S-EPMC3431587 | biostudies-literature | 2012 May-Jun

REPOSITORIES: biostudies-literature

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Selective fluorophore-assisted light inactivation of voltage-gated calcium channels.

Kobrinsky Evgeny E Lee Jung-Ha JH Soldatov Nikolai M NM

Channels (Austin, Tex.) 20120501 3

Fluorophore-assisted light inactivation (FALI) is an investigative tool to inactivate fluorescently labeled proteins by a mechanism of in situ photodestruction. We found that Ca(v)1.2 (L-type) and Ca(v)3.1 (T-type) calcium channels, labeled by genetic fusion with GFP derivatives, show differential sensitivity to FALI. Specifically, FALI silences Ca(v)1.2 calcium channels containing EYFP-labeled α(1C)subunits but does not affect the EYFP-α(1G) Ca(v)3.1 calcium channels or Ca(v)1.2 channels contai ...[more]

PMID: 22909954

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Project description:Propofol is widely used in the clinic for the induction and maintenance of general anesthesia. As with most general anesthetics, however, our understanding of its mechanism of action remains incomplete. Local and general anesthetics largely inhibit voltage-gated Na+ channels (Navs) by inducing an apparent stabilization of the inactivated state, associated in some instances with pore block. To determine the biophysical and molecular basis of propofol action in Navs, we investigated NaChBac and NavMs, two prokaryotic Navs with distinct voltage dependencies and gating kinetics, by whole-cell patch clamp electrophysiology in the absence and presence of propofol at clinically relevant concentrations (2-10 µM). In both Navs, propofol induced a hyperpolarizing shift of the pre-pulse inactivation curve without any significant effects on recovery from inactivation at strongly hyperpolarized voltages, demonstrating that propofol does not stabilize the inactivated state. Moreover, there was no evidence of fast or slow pore block by propofol in a non-inactivating NaChBac mutant (T220A). Propofol also induced hyperpolarizing shifts of the conductance-voltage relationships with negligible effects on the time constants of deactivation at hyperpolarized voltages, indicating that propofol does not stabilize the open state. Instead, propofol decreases the time constants of macroscopic activation and inactivation. Adopting a kinetic scheme of Nav gating that assumes preferential closed-state recovery from inactivation, a 1.7-fold acceleration of the rate constant of activation and a 1.4-fold acceleration of the rate constant of inactivation were sufficient to reproduce experimental observations with computer simulations. In addition, molecular dynamics simulations and molecular docking suggest that propofol binding involves interactions with gating machinery in the S4-S5 linker and external pore regions. Our findings show that propofol is primarily a positive gating modulator of prokaryotic Navs, which ultimately inhibits the channels by promoting activation-coupled inactivation.

Dataset Information

Selective fluorophore-assisted light inactivation of voltage-gated calcium channels.

Publications

Selective fluorophore-assisted light inactivation of voltage-gated calcium channels.

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