Abstract
Pacemaker (HCN) channels have a key role in the generation and modulation of spontaneous activity of sinoatrial node myocytes. Previous work has shown that compartmentation of HCN4 pacemaker channels within caveolae regulates important functions, but the molecular mechanism responsible is still unknown. HCN channels have a conserved caveolin-binding domain (CBD) composed of three aromatic amino acids at the N-terminus; we sought to evaluate the role of this CBD in channel-protein interaction by mutational analysis. We generated two HCN4 mutants with a disrupted CBD (Y259S, F262V) and two with conservative mutations (Y259F, F262Y). In CHO cells expressing endogenous caveolin-1 (cav-1), alteration of the CBD shifted channels activation to more positive potentials, slowed deactivation and made Y259S and F262V mutants insensitive to cholesterol depletion-induced caveolar disorganization. CBD alteration also caused a significant decrease of current density, due to a weaker HCN4-cav-1 interaction and accumulation of cytoplasmic channels. These effects were absent in mutants with a preserved CBD. In caveolin-1-free fibroblasts, HCN4 trafficking was impaired and current density reduced with all constructs; the activation curve of F262V was not altered relative to wt, and that of Y259S displayed only half the shift than in CHO cells. The conserved CBD present in all HCN isoforms mediates their functional interaction with caveolins. The elucidation of the molecular details of HCN4-cav-1 interaction can provide novel information to understand the basis of cardiac phenotypes associated with some forms of caveolinopathies.
| Original language | English |
|---|---|
| Pages (from-to) | 187-195 |
| Number of pages | 9 |
| Journal | Journal of Molecular and Cellular Cardiology |
| Volume | 53 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Aug 2012 |
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Keywords
- Caveolae
- Caveolin binding domain
- HCN
- Pacemaker channels
- Subcellular localization
- Trafficking
ASJC Scopus subject areas
- Molecular Biology
- Cardiology and Cardiovascular Medicine
Cite this
A caveolin-binding domain in the HCN4 channels mediates functional interaction with caveolin proteins. / Barbuti, Andrea; Scavone, Angela; Mazzocchi, Nausicaa; Terragni, Benedetta; Baruscotti, Mirko; DiFrancesco, Dario.
In: Journal of Molecular and Cellular Cardiology, Vol. 53, No. 2, 08.2012, p. 187-195.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A caveolin-binding domain in the HCN4 channels mediates functional interaction with caveolin proteins
AU - Barbuti, Andrea
AU - Scavone, Angela
AU - Mazzocchi, Nausicaa
AU - Terragni, Benedetta
AU - Baruscotti, Mirko
AU - DiFrancesco, Dario
PY - 2012/8
Y1 - 2012/8
N2 - Pacemaker (HCN) channels have a key role in the generation and modulation of spontaneous activity of sinoatrial node myocytes. Previous work has shown that compartmentation of HCN4 pacemaker channels within caveolae regulates important functions, but the molecular mechanism responsible is still unknown. HCN channels have a conserved caveolin-binding domain (CBD) composed of three aromatic amino acids at the N-terminus; we sought to evaluate the role of this CBD in channel-protein interaction by mutational analysis. We generated two HCN4 mutants with a disrupted CBD (Y259S, F262V) and two with conservative mutations (Y259F, F262Y). In CHO cells expressing endogenous caveolin-1 (cav-1), alteration of the CBD shifted channels activation to more positive potentials, slowed deactivation and made Y259S and F262V mutants insensitive to cholesterol depletion-induced caveolar disorganization. CBD alteration also caused a significant decrease of current density, due to a weaker HCN4-cav-1 interaction and accumulation of cytoplasmic channels. These effects were absent in mutants with a preserved CBD. In caveolin-1-free fibroblasts, HCN4 trafficking was impaired and current density reduced with all constructs; the activation curve of F262V was not altered relative to wt, and that of Y259S displayed only half the shift than in CHO cells. The conserved CBD present in all HCN isoforms mediates their functional interaction with caveolins. The elucidation of the molecular details of HCN4-cav-1 interaction can provide novel information to understand the basis of cardiac phenotypes associated with some forms of caveolinopathies.
AB - Pacemaker (HCN) channels have a key role in the generation and modulation of spontaneous activity of sinoatrial node myocytes. Previous work has shown that compartmentation of HCN4 pacemaker channels within caveolae regulates important functions, but the molecular mechanism responsible is still unknown. HCN channels have a conserved caveolin-binding domain (CBD) composed of three aromatic amino acids at the N-terminus; we sought to evaluate the role of this CBD in channel-protein interaction by mutational analysis. We generated two HCN4 mutants with a disrupted CBD (Y259S, F262V) and two with conservative mutations (Y259F, F262Y). In CHO cells expressing endogenous caveolin-1 (cav-1), alteration of the CBD shifted channels activation to more positive potentials, slowed deactivation and made Y259S and F262V mutants insensitive to cholesterol depletion-induced caveolar disorganization. CBD alteration also caused a significant decrease of current density, due to a weaker HCN4-cav-1 interaction and accumulation of cytoplasmic channels. These effects were absent in mutants with a preserved CBD. In caveolin-1-free fibroblasts, HCN4 trafficking was impaired and current density reduced with all constructs; the activation curve of F262V was not altered relative to wt, and that of Y259S displayed only half the shift than in CHO cells. The conserved CBD present in all HCN isoforms mediates their functional interaction with caveolins. The elucidation of the molecular details of HCN4-cav-1 interaction can provide novel information to understand the basis of cardiac phenotypes associated with some forms of caveolinopathies.
KW - Caveolae
KW - Caveolin binding domain
KW - HCN
KW - Pacemaker channels
KW - Subcellular localization
KW - Trafficking
UR - http://www.scopus.com/inward/record.url?scp=84862872728&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84862872728&partnerID=8YFLogxK
U2 - 10.1016/j.yjmcc.2012.05.013
DO - 10.1016/j.yjmcc.2012.05.013
M3 - Article
C2 - 22659290
AN - SCOPUS:84862872728
VL - 53
SP - 187
EP - 195
JO - Journal of Molecular and Cellular Cardiology
JF - Journal of Molecular and Cellular Cardiology
SN - 0022-2828
IS - 2
ER -