Regulation of cell surface expression of functional pacemaker channels by a motif in the B-helix of the cyclic nucleotide-binding domain

Nazzari, Hamed; Angoli, Damiano; Chow, Sarah S.; Whitaker, Gina M.; Leclair, Leisha; McDonald, Evan; Macri, Vincenzo; Zahynacz, Kristin; Walker, Valerie E.; Accili, Eric A.

doi:10.1152/ajpcell.00062.2008

Cited by 5 publications

(6 citation statements)

References 33 publications

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“…Recently, a four-amino acid motif (EEYP) in the B-helix of HCN2 was identified that strongly promotes channel export from the endoplasmatic reticulum and targeting to the cell membrane (287).…”

Section: B Pore Loop and Selectivity Filtermentioning

confidence: 99%

Hyperpolarization-Activated Cation Channels: From Genes to Function

Biel

Wahl‐Schott²,

Michalakis³

et al. 2009

Physiological Reviews

872

974

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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels comprise a small subfamily of proteins within the superfamily of pore-loop cation channels. In mammals, the HCN channel family comprises four members (HCN1-4) that are expressed in heart and nervous system. The current produced by HCN channels has been known as Ih (or If or Iq). Ih has also been designated as pacemaker current, because it plays a key role in controlling rhythmic activity of cardiac pacemaker cells and spontaneously firing neurons. Extensive studies over the last decade have provided convincing evidence that Ih is also involved in a number of basic physiological processes that are not directly associated with rhythmicity. Examples for these non-pacemaking functions of Ih are the determination of the resting membrane potential, dendritic integration, synaptic transmission, and learning. In this review we summarize recent insights into the structure, function, and cellular regulation of HCN channels. We also discuss in detail the different aspects of HCN channel physiology in the heart and nervous system. To this end, evidence on the role of individual HCN channel types arising from the analysis of HCN knockout mouse models is discussed. Finally, we provide an overview of the impact of HCN channels on the pathogenesis of several diseases and discuss recent attempts to establish HCN channels as drug targets.

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Section: B Pore Loop and Selectivity Filtermentioning

confidence: 99%

Hyperpolarization-Activated Cation Channels: From Genes to Function

Biel

Wahl‐Schott²,

Michalakis³

et al. 2009

Physiological Reviews

872

974

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“…This system was chosen for two reasons: first, nonglycosylated Shaker, the prototypical channel in the Kv superfamily, traffics to the plasma membrane much more efficiently in oocytes than in HEK cells (5,38). Second, certain HCN2 trafficking mutants that do not express functionally in mammalian cells (33,35) do so in oocytes (49). For comparison, we also expressed mHCN2-N380Q or wild-type mHCN2 in CHO cells.…”

Section: Hcn2 Channels Undergo N-glycosylation In Native Cardiacmentioning

confidence: 99%

Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression

Hegle

Nazzari

Roth

et al. 2010

American Journal of Physiology-Cell Physiology

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Hegle AP, Nazzari H, Roth A, Angoli D, Accili EA. Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression. Am J Physiol Cell Physiol 298: C1066 -C1076, 2010. First published February 3, 2010 doi:10.1152/ajpcell.00389.2009.-All four mammalian hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel isoforms have been shown to undergo N-linked glycosylation in the brain. With the mouse HCN2 isoform as a prototype, HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. This difference between HCN1 and HCN2 is consistent with evolutionary trajectories that diverged in an isoform-specific manner after gene duplication from a common HCN ancestor that lacked N-glycosylation and was able to localize efficiently to the cell surface. ion channels; hyperpolarization-activated cyclic nucleotide-modulated channel-mediated current; N-linked glycosylation; embryonic heart; molecular evolution AN OUTSTANDING QUESTION in hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel biology, and indeed in the biology of most intrinsic proteins of the plasma membrane, is how expression at the cell surface is regulated to affect functional heterogeneity in health and pathological states. The number of HCN channels on the cell surface is a critical determinant of beating frequency in cardiac conduction tissue. HCN isoforms and HCN-mediated currents (I h ) are upregulated in the embryonic and neonatal ventricle (24,45,53,56), in the neonatal sinoatrial node (1), and in beating embryonic stem cells during development in culture (27,36,40,41). However, the factors that determine HCN channel supply at the cell surface during cardiac development have been studied in only a limited fashion.An important determinant of plasma membrane expression of intrinsic membrane proteins is N-linked glycosylation, which promotes proper folding, stability, and oligomeric assembly in the endoplasmic reticulum (ER) and facilitates transport and targeting to the plasma membrane (14, 15). HCN1 and HCN2 are extensively N-glycosylated in mouse brain (31,39,58) and contain only one Asn-X-Ser/Thr consensus sequon for N-glycosylation in the S5 linker, close to the channel pore (Fig.

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“…Together these data suggested that the doublet represented the GFP-HCN2 channel. Previous studies on HCN2 channels expressed in Hek cells showed that the doublet represented two forms of the HCN2 channel (Much et al, 2003; Akhavan et al, 2005; Nazzari et al, 2008). The slower migrating band contained a complex N -glycosylation.…”

Section: Resultsmentioning

confidence: 99%

“…The faster migrating band instead possessed a simple or no glycosylation. Complex N -glycosylation increased the efficiency of surface expression, but channels possessing only a subset of complex N -glycosylated subunits (Akhavan et al, 2005) or no complex N -glycosylated subunits (Nazzari et al, 2008) could still be detected at the plasma membrane. Relative to past studies, the doublet detected in our experiments is larger, presumably due to the GFP tag.…”

Section: Resultsmentioning

confidence: 99%

“…Other interaction motifs also exist (Flotho and Melchior, 2013). We have demonstrated that HCN2 channel SUMOylation can regulate surface expression, and that (putative) HCN2 SUMOylation sites are located in domains that are important for channel trafficking and surface expression (Proenza et al, 2002; Tran et al, 2002; Akhavan et al, 2005; Nazzari et al, 2008). Thus, while SIM containing interaction partners for HCN2 channels have not yet been identified, it seems likely that they may include trafficking and scaffold proteins.…”

Section: Discussionmentioning

confidence: 99%

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SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

Parker

Welch

Forster

et al. 2017

Front. Mol. Neurosci.

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Small Ubiquitin-like Modifier (SUMO) is a ∼10 kDa peptide that can be post-translationally added to a lysine (K) on a target protein to facilitate protein–protein interactions. Recent studies have found that SUMOylation can be regulated in an activity-dependent manner and that ion channel SUMOylation can alter the biophysical properties and surface expression of the channel. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel surface expression can be regulated in an activity-dependent manner through unknown processes. We hypothesized that SUMOylation might influence the surface expression of HCN2 channels. In this manuscript, we show that HCN2 channels are SUMOylated in the mouse brain. Baseline levels of SUMOylation were also observed for a GFP-tagged HCN2 channel stably expressed in Human embryonic kidney (Hek) cells. Elevating GFP-HCN2 channel SUMOylation above baseline in Hek cells led to an increase in surface expression that augmented the hyperpolarization-activated current (Ih) mediated by these channels. Increased SUMOylation did not alter Ih voltage-dependence or kinetics of activation. There are five predicted intracellular SUMOylation sites on HCN2. Site-directed mutagenesis indicated that more than one K on the GFP-HCN2 channel was SUMOylated. Enhancing SUMOylation at one of the five predicted sites, K669, led to the increase in surface expression and Ih Gmax. The role of SUMOylation at additional sites is currently unknown. The SUMOylation site at K669 is also conserved in HCN1 channels. Aberrant SUMOylation has been linked to neurological diseases that also display alterations in HCN1 and HCN2 channel expression, such as seizures and Parkinson’s disease. This work is the first report that HCN channels can be SUMOylated and that this can regulate surface expression and Ih.

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Regulation of cell surface expression of functional pacemaker channels by a motif in the B-helix of the cyclic nucleotide-binding domain

Cited by 5 publications

References 33 publications

Hyperpolarization-Activated Cation Channels: From Genes to Function

Hyperpolarization-Activated Cation Channels: From Genes to Function

Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression

SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

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