Proteolytic Processing of HCN2 and Co-assembly with HCN4 in the Generation of Cardiac Pacemaker Channels

Abstract

In sino-atrial and atrio-ventricular nodal cells, hyperpolarization-activated cyclic nucleotide-gated (HCN) inward current carrying cationic channels, I f , are expressed that contribute importantly to the diastolic depolarization critical for cardiac pacemaker activity. Although previous studies have demonstrated myocardial expression of both the HCN2 and HCN4 subunits, the specific roles of these subunits in the generation of functional myocardial I f channels remain unclear. To explore the molecular compositions of functional cardiac I f channels, antibodies targeted against specific C-and N-terminal sequences in HCN2 and HCN4 were exploited to examine HCN2 and HCN4 subunit expression in adult (mouse) heart and to immunoprecipitate endogenous HCN-encoded cardiac I f channel complexes. Western blot experiments revealed that although the full-length HCN2 (105 kDa) and HCN4 (160 kDa) proteins are readily detected in transiently transfected HEK-293 cells and in adult (mouse) brain, the molecular mass of the HCN2 protein in the myocardium is ϳ60 kDa. In addition, the myocardial 60-kDa HCN2 protein lacks the C terminus, which contains the cAMP binding domain. In heterologous cells, the C-terminal-truncated HCN2 protein co-assembles with HCN4 to form functional heteromeric HCN channels, which activate faster than homomeric HCN2 or homomeric HCN4 channels, and display properties similar to endogenous myocardial I f channels Taken together, these results suggest that functional myocardial I f channels reflect the heteromeric assembly of HCN2 and HCN4 subunits and further that the HCN4 subunit underlies the cAMP-mediated regulation of cardiac I f channels.Hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 cationic currents are responsible for generating spontaneous pacemaker potentials in the heart (1-3) and in the central nervous system (4). Four members of the HCN family, HCN1-4, have been identified and extensively characterized in heterologous cells (5-9). Similar to voltage-gated K ϩ (Kv) channels, each HCN channel subunit contains six transmembrane domains and a pore region with a GYG signature motif (5, 6). Heterologous expression of the various HCN subunits reveals hyperpolarization-activated cationic inward currents with distinct voltage-and time-dependent properties, as well as differential sensitivities to cAMPdependent modulation (8, 9). In the myocardium, HCN4 is the most abundantly expressed transcript (5), whereas HCN1 is the primary HCN transcript in brain (5) and HCN2 is readily detected in both heart and brain (7). Previous studies have shown that the conserved N-terminal 52-amino acid sequence in each of the HCN proteins plays critical roles in channel assembly and trafficking (10). In addition, there is a cyclic nucleotide binding domain (120 amino acids) in the C termini of the HCN subunits (8,11,12), although the C-terminal amino acid sequences of each (HCN1-4) of the HCN proteins are distinct.Although the molecular compositions of functional cardiac and neuronal I f channels have not bee...

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

“…In the presence of PNGaseF, the HCN2 band is completely shifted to ϳ90 kDa, suggesting that cardiac HCN2 channels are fully N-glycosylated in vivo. These molecular masses are consistent with a nonproteolyzed form of HCN2, as has been reported for rat and human ventricles (17,36,46,57,59).…”

HCN Channel C-Terminal Region Speeds Activation Rates Independently of Autoinhibition

“…Insufficiency of Simple Autoinhibition Model Based on V 1/2 Assessment Consequences for HCN channel modulation from truncations of the C-terminal region are likely to have physiological relevance, since C-terminal truncations have been identified in human patients with cardiac sinus node defects (Schulze-Bahr et al 2003;Schweizer et al 2010) and even in myocardial tissues of healthy mice (Ye and Nerbonne 2009). Thus, it is essential to generate a cohesive biophysical theory of how the molecular structure can determine kinetic properties.…”

Expression and distribution of voltage-gated ion channels in ferret sinoatrial node

“…3) (22,28,41,45,67,69,71,80,84). Comparison of transcripts from all four hyperpolarization-activated cation channels showed that expression of HCN1 and HCN4 were highest, although there were significant levels of HCN2 and HCN3 (Fig.…”