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 retinal L-type Ca 2؉ channel Cav1.4 is distinguished from all other members of the high voltage-activated (HVA) Ca 2؉ channel family by lacking Ca 2؉ -calmodulin-dependent inactivation. In synaptic terminals of photoreceptors and bipolar cells, this feature is essential to translate graded membrane depolarizations into sustained Ca 2؉ influx and tonic glutamate release. The sequences conferring Ca 2؉ -dependent inactivation (CDI) are conserved throughout the HVA calcium channel family, raising the question of how Cav1.4 manages to switch off CDI. Here, we identify an autoinhibitory domain in the distal C terminus of Cav1.4 that serves to abolish CDI. We show that this domain (ICDI, inhibitor of CDI) uncouples the molecular machinery conferring CDI from the inactivation gate by binding to the EF hand motif in the proximal C terminus. Deletion of ICDI completely restores Ca 2؉ -calmodulinmediated CDI in Cav1.4. CDI can be switched off again in the truncated Cav1.4 channel by coexpression of ICDI, indicating that ICDI works as an autonomous unit. Furthermore, we show that in the Cav1.2 L-type Ca 2؉ -channel replacement of the distal C terminus by the corresponding sequence of Cav1.4 is sufficient to block CDI. This finding suggests that autoinhibition of CDI can be introduced principally into other Ca 2؉ channel types. Our data provide a previously undescribed perspective on the regulation of HVA calcium channels by Ca 2؉ .calmodulin ͉ Cav1.4 ͉ retina ͉ Ca channels

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr 476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr 476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.

The data indicate that Ca(v)1.4alpha1 subunit constitutes the major molecular correlate of retinal L-type Ca(2+) current. Its intrinsic biophysical properties, in particular its unique inactivation properties, enable Ca(v)1.4alpha1 to provide a sustained I(Ca) over a voltage range such as required for tonic glutamate release at the photoreceptor synapse.

The modulation of ion channel activity by extracellular ions plays a central role in the control of heart function. Here, we show that the sinoatrial pacemaker current I f is strongly affected by the extracellular Cl ؊ concentration. We investigated the molecular basis of the Cl ؊ dependence in heterologously expressed hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that represent the molecular correlate of I f . Currents carried by the two cardiac HCN channel isoforms (HCN2 and HCN4) showed the same strong Cl ؊ dependence as the sinoatrial I f and decreased to about 10% in the absence of external Cl ؊ . In contrast, the neuronal HCN1 current was reduced to only 50% under the same conditions. Depletion of Cl ؊ did not affect the voltage dependence of activation or the ion selectivity of the channels, indicating that the reduction of I f was caused by a decrease of channel conductance. A series of chimeras between HCN1 and HCN2 was constructed to identify the structural determinants underlying the different Cl ؊ dependence of HCN1 and HCN2. Exchange of the ion-conducting pore region was sufficient to switch the Cl ؊ dependence from HCN1-to HCN2-type and vice versa. Replacement of a single alanine residue in the pore of HCN1 (Ala-352) by an arginine residue present in HCN2 at equivalent position (Arg-405) induced HCN2-type chloride sensitivity in HCN1. Our data indicate that Arg-405 is a key component of a domain that allosterically couples Cl ؊ binding with channel activation.The slow diastolic depolarization in sinoatrial node (SAN) 1 cells is the motor of cellular automaticity that drives the spontaneous beating of the heart (1-3). In addition, the slow diastolic depolarization is in the center of the autonomous control of heart rate because its kinetics is speeded up or slowed down by the release of norepinephrine and acetylcholine from sympathetic and parasympathetic nerve terminals, respectively. Several ionic currents have been proposed to control the time course and slope of slow diastolic depolarizations (e.g. T-type and L-type Ca 2ϩ currents and the sustained inward current I st ), some of which are directly regulated by the autonomous nervous system (4). Among these currents, the hyperpolarization-activated ("pacemaker") current I f plays an outstanding role. As the only current in SAN node, I f is dually gated by both hyperpolarized voltages and binding of cAMP, a second messenger, the concentration of which is directly controlled by the activity of ␤-adrenergic and muscarinic receptors. Owing to its dual gating behavior, I f contributes both to basal heart rate and to the autonomous regulation of rhythmicity. I f is carried by a family of four hyperpolarization activated-and cyclic nucleotide-gated channels (HCN1-4) (1, 5). Genetic studies in human patients suffering from sick sinus syndrome (6, 7) and the analysis of HCN4-deficient mice (8) indicated that the HCN4 channel is required for normal sympathetic stimulation of pacemaker activity. In contrast, HCN2 is not involved in auto...