Abstract
Sarcoplasmic reticulum (SR) vesicles were prepared from either canine
or sheep heart and fused into lipid bilayers to study their ionic
channels. A 92 +/- 5 pS anion-selective channel was recorded in asymmetric
50 mM trans/250 mM cis CsCl buffer system. Reversal potentials and
theoretical equilibrium potentials for Cl$^-$ions obtained under
various experimental conditions allowed us to confirm the Cl$^-$
selectivity of this SR channel. The majority (69\%) of channel recordings
(n = 45) displayed steady-state kinetics and a slight voltage dependency
of the open probability. However, 31\% of the channels inactivated
after their incorporation. We now report that the channel might be
reactivated by depolarizing voltage steps. Furthermore, the use of
either PKA or PKG in association with adequate phosphorylating
buffers lengthens the deactivation process at the end of the voltage
pulses, but does not prevent the inactivation. It was assumed that
the change in gating mode was due to a voltage-sensitive association/dissociation
mechanism with a phosphorylated protein of the SR membrane such as
phospholamban (PL). We demonstrated that a specific monoclonal
antibody raised against canine PL inhibited the activity of the
channel and prevented its reactivation by depolarizing steps. 400
to 800 ng/ml of Anti-PL Ab consistently and sequentially turned
off the channel activities. In contrast, heat inactivated Anti-PL
Ab had no effect. We propose that phospholamban may be a primer of
the SR Cl$^-$ channel whereby Cl$^-$ anions would play the
role of counter-charge carrier during rapid Ca$^2+$ release and
Ca$^2+$ uptake by the SR.
- 8568846
- animals,
- antibodies,
- bilayers,
- calcium-binding
- channel
- channels,
- chloride
- chlorides,
- dogs,
- gating,
- gov't,
- ion
- lipid
- membrane
- monoclonal,
- muscle
- myocardium,
- non-u.s.
- phosphorylation,
- post-translational,
- potentials,
- processing,
- protein
- proteins,
- research
- reticulum,
- sarcoplasmic
- sheep,
- support,
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