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...

The pacemaker channels HCN2 and HCN4 have been identified in cardiac sino-atrial node cells. These channels differ considerably in several kinetic properties including the activation time constant ( act ), which is fast for HCN2 (144 ms at ؊140 mV) and slow for HCN4 (461 ms at ؊140 mV). Here, by analyzing HCN2/4 chimeras and mutants we identified single amino acid residues in transmembrane segments 1 and 2 and the connecting loop between S1 and S2 that are major determinants of this difference. Replacement of leucine 272 in S1 of HCN4 by the corresponding phenylalanine present in HCN2 decreased act of HCN4 to 149 ms. Conversely, activation of the fast channel HCN2 was decreased 3-fold upon the corresponding mutation of F221L in the S1 segment. Mutation of N291T and T293A in the linker between S1 and S2 of HCN4 shifted act to 275 ms. While residues 272, 291, and 293 of HCN4 affected the activation speed at basal conditions they had no obvious influence on the cAMP-dependent acceleration of activation kinetics. In contrast, mutation of I308M in S2 of HCN4 abolished the cAMP-dependent decrease in act . Surprisingly, this mutation also prevented the acceleration of channel activation observed after deletion of the C-terminal cAMP binding site. Taken together our results indicate that the speed of activation of the HCN4 channel is determined by structural elements present in the S1, S1-S2 linker, and the S2 segment.Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) 1 channels are thought to underlie the native pacemaker current, termed I f or I h , in the heart and brain where it contributes to the rhythmic activity of cardiac and neuronal pacemaker cells (1-4). All four members of the mammalian HCN channel gene family that have been cloned recently (5-9) share a highly preserved core region containing 6 transmembrane segments, including a voltage-sensing S4 segment and a pore region between S5 and S6, which is homologous to the S1-S6 region of voltage-gated potassium channels (K v ). The homology of the intracellular N and C termini within the HCN subtypes is less pronounced than for the core region with the exception of the highly conserved 120 amino acid long cyclic nucleotide binding domain (CNBD) starting about 80 amino acids downstream of S6.All four HCN channels are expressed in cardiac tissue (5, 10), but with some variations in the expression intensities among different species. HCN4 is highly expressed in the sino-atrial node. It is generally assumed that the slowly activating HCN4 contributes to the pacemaker activity and the modulation of the heart rate by ␤-adrenergic stimulation, whereas the less expressed, faster activated HCN2 and HCN1 may have additional functions such as maintaining the resting potential of pacemaker and other cells (11)(12)(13)(14).All four HCN channels are activated upon membrane hyperpolarization. Activation is voltage-dependent, i.e. the more hyperpolarized the membrane becomes, the faster the channels open. The HCN channels differ, however, greatly in their activ...

A DNA-based approach allows external control over the self-assembly process of tobacco mosaic virus (TMV)-like ribonucleoprotein nanotubes: their growth from viral coat protein (CP) subunits on five distinct RNA scaffolds containing the TMV origin of assembly (OAs) could be temporarily blocked by a stopper DNA oligomer hybridized downstream (3') of the OAs. At two upstream (5') sites tested, simple hybridization was not sufficient for stable stalling, which correlates with previous findings on a non-symmetric assembly of TMV. The growth of DNA-arrested particles could be restarted efficiently by displacement of the stopper via its toehold by using a release DNA oligomer, even after storage for twelve days. This novel strategy for growing proteinaceous tubes under tight kinetic and spatial control combines RNA guidance and its site-specific but reversible interruption by DNA blocking elements. As three of the RNA scaffolds contained long heterologous non-TMV sequence portions that included the stopping sites, this method is applicable to all RNAs amenable to TMV CP encapsidation, albeit with variable efficiency most likely depending on the scaffolds' secondary structures. The use of two distinct, selectively addressable CP variants during the serial assembly stages finally enabled an externally configured fabrication of nanotubes with highly defined subdomains. The "stop-and-go" strategy thus might pave the way towards production routines of TMV-like particles with variable aspect ratios from a single RNA scaffold, and of nanotubes with two or even more adjacent protein domains of tightly pre-defined lengths.

The pea mutant line P55 is defective in root nodule formation, and this phenotype is controlled by a single recessive gene. Complementation analysis revealed that the mutation in P55 is allelic to sym19, which has previously been mapped to linkage group I. Detailed mapping revealed that the sym19 and ENOD40 loci are separated by 2.7 cM. We identified four recombination events, demonstrating that the nodulation defect caused by mutation of the sym19 locus cannot be due to mutation of ENOD40. RT-PCR experiments showed that P55 expresses ENOD12A, but there was little or no increase in the level of its transcript in response to Nod factor or infection with Rhizobium. To investigate this expression pattern further, transgenic peas carrying a pENOD12A-GUS reporter construct were made. One transgenic line was crossed with line P55, to generate F2 progeny homozygous for sym19 and carrying pENOD12A-GUS. In both WT and sym19 mutant lines, ENOD12A-GUS expression was induced at sites of lateral root emergence in uninoculated plants. In Nod+ plants pENOD12A-GUS was induced in response to Rhizobium leguminosarumn bv. viciae, but no such induction was seen in the Nod- (sym19) mutants.

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