Voltage-gated rearrangements associated with differential beta-subunit modulation of the L-type Ca(2+) channel inactivation.
Auxiliary beta-subunits bound to the cytoplasmic alpha(1)-interaction domain of the pore-forming alpha(1C)-subunit are important modulators of voltage-gated Ca(2+) channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by beta(1a)- and beta(2)- subunits and structural responses of the channel to transition into distinct functional states. The NH(2)-termini of the alpha(1C)- and beta-subunits were fused with cyan or yellow fluorescent proteins, and functionally coexpressed in COS1 cells. Fluorescence resonance energy transfer (FRET) between them or with membrane-trapped probes was measured in live cells under voltage clamp. It was found that in the resting state, the tagged NH(2)-termini of the alpha(1C)- and beta-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between alpha(1C) and beta(1a) suggesting mutual reorientation of the NH(2)-termini, but their distance vis-à-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the beta(1a)-subunit was replaced by beta(2). Differential beta-subunit modulation of inactivation and of FRET between alpha(1C) and beta were eliminated by inhibition of the slow inactivation. Thus, differential beta-subunit modulation of inactivation correlates with the voltage-gated motion between the NH(2)-termini of alpha(1C)- and beta-subunits and targets the mechanism of slow voltage-dependent inactivation.
Kobrinsky
Evgeny
E
National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
Kepplinger
Klaus J F
KJ
Yu
Alexander
A
Harry
Jo Beth
JB
Kahr
Heike
H
Romanin
Christoph
C
Abernethy
Darrell R
DR
Soldatov
Nikolai M
NM
D000818:Animals; D019556:COS Cells; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007668:Kidney / Q000737:chemistry / Q000196:embryology / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013223:Statistics as Topic; D013329:Structure-Activity Relationship
D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D021122:Protein Subunits; D011994:Recombinant Proteins