Role of Subunit Heteromerization and N-Linked Glycosylation in the Formation of Functional Hyperpolarization-activated Cyclic Nucleotide-gated Channels

Abstract

The coassembly of homologous subunits to heteromeric complexes serves as an important mechanism in generating ion channel diversity. Here, we have studied heteromerization in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family. Using a combination of fluorescence confocal microscopy, coimmunoprecipitation, and electrophysiology we found that upon coexpression in HEK293 cells almost all dimeric combinations of HCN channel subunits give rise to the formation of stable channel complexes in the plasma membrane. We also identified HCN1/HCN2 heteromers in mouse brain indicating that heteromeric channels exist in vivo. Surprisingly, HCN2 and HCN3 did not coassemble to heteromeric channels. This finding indicates that heteromerization requires specific structural determinants that are not present in all HCN channel combinations. Using N-glycosidase F we show that native as well as recombinant HCN channels are glycosylated resulting in a 10 -20-kDa shift in the molecular weight. Tunicamycin, an inhibitor of Nlinked glycosylation, blocked surface membrane expression of HCN2. Similarly, a mutant HCN2 channel in which the putative N-glycosylation site in the loop between S5 and the pore helix was replaced by glutamine (HCN2 N380Q ) was not inserted into the plasma membrane and did not yield detectable whole-cell currents. These results indicate that N-linked glycosylation is required for cell surface trafficking of HCN channels. Cotransfection of HCN2 N380Q with HCN4, but not with HCN3, rescued cell surface expression of HCN2 N380Q . Immunoprecipitation revealed that this rescue was due to the formation of a HCN2 N380Q /HCN4 heteromeric channel. Taken together our results indicate that subunit heteromerization and glycosylation are important determinants of the formation of native HCN channels.The hyperpolarization-activated cation current I h (or I f, I q ) plays a key role in the control of important biological processes such as heart beat (1), sleep-wake cycle (2), transduction of sour taste (3), and synaptic plasticity (4). I h is encoded by the hyperpolarization-activated cyclic nucleotide-gated (HCN) 1 channel gene family (for review, see Refs. 5-7). In mammals, the HCN channel family comprises four homologous members (HCN1-4). Structurally, HCN channels belong to the superfamily of voltage-gated cation channels. Like other members of this family HCN channels are supposed to form tetramers with fundamental building blocks consisting of six hydrophobic segments (S1-S6), a positively charged S4 sensor, and an ionconducting hairpin between S5 and S6. In the cytosolic carboxyl terminus each of the four HCN channel subunits carries a cyclic nucleotide-binding site mediating modulation by cAMP. Expression of HCN1-4 cDNAs in heterologous expression systems yields currents with the hallmark properties of I h , namely activation by membrane hyperpolarization, permeation of Na ϩ and K ϩ , shift of the activation curve to more depolarized voltages by intracellular binding of cAMP, and blockage by low mil...

PIP2-Mediated HCN3 Channel Gating Is Crucial for Rhythmic Burst Firing in Thalamic Intergeniculate Leaflet Neurons

“…2, 3). Third, HCN2/3 heteromers do not appear to exist (Much et al, 2003). Thus, our I h data indicate that no cAMP-sensitive homomeric HCN2 channels are involved, and are in keeping with the observed biophysical properties of recombinant HCN3 channels, which feature slow activation kinetics and no facilitatory gating effect during cAMP application (Mistrík et al, 2005;Stieber et al, 2005).…”

Formation of heteromeric hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the hippocampus is regulated by developmental seizures

“…While the ability of HCN1 and HCN2 to co-assemble into functional channels has been demonstrated (Chen et al, 2001b;Er et al, 2003;Much et al, 2003;Proenza et al, 2002;Ulens and Tytgat, 2001;Xue et al, 2002), we find only modest co-association of HCN1/HCN2 in the 'normal' hippocampal formation of both developing and adult rats (control groups, Figs. 1 and 5), consistent with the single report on whole mouse brain (Much et al, 2003). However and importantly, we demonstrate that this apparent heteromerization is a dynamic, malleable process that is governed by network activity.…”

“…1C, top). In contrast, IP with anti-HCN2 followed by probing for HCN1 yielded a protein band with the expected molecular weight of ∼112 kDa (Much et al, 2003) and higher expression levels in hippocampus (Fig. 1C, bottom).…”

“…By contrast, homomeric HCN2 channels conduct I h currents with slower kinetics and robust cAMP-evoked shifts in voltage dependence (Ludwig et al, 1998;Robinson and Siegelbaum, 2003). More recently, the formation of heteromeric channels in vitro (Chen et al, 2001b;Er et al, 2003;Much et al, 2003;Proenza et al, 2002;Ulens and Tytgat, 2001;Xue et al, 2002) as well as in vivo (Er et al, 2003;Much et al, 2003) has been described. However, the mechanisms promoting heteromerization have remained unclear.…”

Impaired Channel Targeting and Retinal Degeneration in Mice Lacking the Cyclic Nucleotide-Gated Channel Subunit CNGB1

“…Membrane proteins were isolated from either murine retinas or transfected HEK293 cells as described previously (Much et al, 2003). Equal amounts of proteins were separated using 7-10% SDS-PAGE, followed by Western blot analysis according to standard procedures.…”