TY - JOUR
T1 - Voltage-gated rearrangements associated with differential β-subunit modulation of the L-type Ca2+ channel inactivation
AU - Kobrinsky, Evgeny
AU - Kepplinger, Klaus J.F.
AU - Yu, Alexander
AU - Harry, Jo Beth
AU - Kahr, Heike
AU - Romanin, Christoph
AU - Abernethy, Darrell R.
AU - Soldatov, Nikolai M.
N1 - Funding Information:
This work was supported in part by the National Institute on Aging Intramural Research Program (N.M.S.) and Austrian Science Foundation (P-15387 to C.R.).
PY - 2004/8
Y1 - 2004/8
N2 - Auxiliary β-subunits bound to the cytoplasmic α1- interaction domain of the pore-forming α1C-subunit are important modulators of voltage-gated Ca2+ channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by β1a- and β2-subunits and structural responses of the channel to transition into distinct functional states. The NH2-termini of the α1C- and β-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 NH2-termini of the α1C- and β-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between α1C and β1a suggesting mutual reorientation of the NH2-termini, but their distance vis-à-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the β1a-subunit was replaced by β2. Differential β-subunit modulation of inactivation and of FRET between α1C and β were eliminated by inhibition of the slow inactivation. Thus, differential β-subunit modulation of inactivation correlates with the voltage-gated motion between the NH2-termini of α1C- and β-subunits and targets the mechanism of slow voltage-dependent inactivation.
AB - Auxiliary β-subunits bound to the cytoplasmic α1- interaction domain of the pore-forming α1C-subunit are important modulators of voltage-gated Ca2+ channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by β1a- and β2-subunits and structural responses of the channel to transition into distinct functional states. The NH2-termini of the α1C- and β-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 NH2-termini of the α1C- and β-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between α1C and β1a suggesting mutual reorientation of the NH2-termini, but their distance vis-à-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the β1a-subunit was replaced by β2. Differential β-subunit modulation of inactivation and of FRET between α1C and β were eliminated by inhibition of the slow inactivation. Thus, differential β-subunit modulation of inactivation correlates with the voltage-gated motion between the NH2-termini of α1C- and β-subunits and targets the mechanism of slow voltage-dependent inactivation.
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U2 - 10.1529/biophysj.104.041152
DO - 10.1529/biophysj.104.041152
M3 - Article
C2 - 15298893
AN - SCOPUS:4143091363
SN - 0006-3495
VL - 87
SP - 844
EP - 857
JO - Biophysical journal
JF - Biophysical journal
IS - 2
ER -