Redox- and Calmodulin-dependent S-Nitrosylation of the KCNQ1 Channel

Ken Asada,Junko Kurokawa,Tetsushi Furukawa

doi:10.1074/jbc.m807158200

Ken Asada, Junko Kurokawa + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m807158200

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Abstract

Nitric oxide (NO) is a gaseous signal mediator showing numerous important biological effects. NO has been shown in many instances to exhibit its action via the protein S-nitrosylation mechanism, in which binding of NO to Cys residues regulate protein function independently of activation of soluble guanylate cyclase. The direct link between protein S-nitrosylation and functional modulation, however, has been demonstrated only in limited examples. Furthermore, although most proteins have more than one Cys residue, the mechanism by which a certain Cys becomes a specific target residue of S-nitrosylation is poorly understood. We have previously reported that NO regulates currents through the cardiac slowly activating delayed rectifier potassium channel (I(Ks)) irrespective of soluble guanylate cyclase activation. Here we demonstrate using a biotin-switch assay that NO induced S-nitrosylation of the alpha-subunit of the I(Ks) channel, KCNQ1, at Cys(445) in the C terminus. A redox motif flanking Cys(445) and the interaction of KCNQ1 with calmodulin are required for preferential S-nitrosylation of Cys(445). A patch clamp experiment shows that S-nitrosylation of Cys(445) modulates the KCNQ1/KCNE1 channel function. Our data provide a molecular basis of NO-mediated regulation of the I(Ks) channel. This novel regulatory mechanism of the I(Ks) channel may play a role in previously demonstrated NO-mediated phenomenon in cardiac electrophysiology, including shortening in action potential duration in response to intracellular Ca(2+) or sex hormones.

Highlights

Verted to a nitrosothiol [1,2,3] and acts as a regulatory mechanism of various classes of proteins, including ion channels, such as the skeletal muscle type ryanodine receptor channel [4, 5], the N-methyl-D-aspartate receptor channel [6, 7], the cardiac L-type Ca2ϩ channel [8], and the cardiac Naϩ channel [9]
The following issues remain to be addressed: (i) Is the IKs channel S-nitrosylated? (ii) If so, what is the target of S-nitrosylation between the ␣-subunit KCNQ1 and the ␤-subunit KCNE1? (iii) Among multiple Cys residues, which Cys is a target of S-nitrosylation? and (iv) How does Nitric oxide (NO) recognize the target Cys? In the present study, we used the biotin-switch assay and functional patch clamp experiment to answer these questions
We confirmed that transfection of HEK293 cells with V5-tagged KCNQ1 and KCNE1 yielded similar currents to those obtained by transfection with KCNQ1 and KCNE1

Summary

Introduction

Verted to a nitrosothiol [1,2,3] and acts as a regulatory mechanism of various classes of proteins, including ion channels, such as the skeletal muscle type ryanodine receptor (ryanodine receptor type 1) channel [4, 5], the N-methyl-D-aspartate receptor channel [6, 7], the cardiac L-type Ca2ϩ channel [8], and the cardiac Naϩ channel [9]. S-Nitrosylation was detected only in the GST fusion protein with Cys381 and Cys445 (Val355– Leu619 of KCNQ1) (lane 3, Fig. 3B).

Results

Conclusion