Abstract-Lipid rafts are discrete membrane subdomains rich in sphingolipids and cholesterol. In ventricular myocytes a function of caveolae, a type of lipid rafts, is to concentrate in close proximity several proteins of the ␤-adrenergic transduction pathway. We have investigated the subcellular localization of HCN4 channels expressed in HEK cells and studied the effects of such localization on the properties of pacemaker channels in HEK and rabbit sinoatrial (SAN) cells. We used a discontinuous sucrose gradient and Western blot analysis to detect HCN4 proteins in HEK and in SAN cells, and found that HCN4 proteins localize to low-density membrane fractions together with flotillin (HEK) or caveolin-3 (SAN), structural proteins of caveolae. Lipid raft disruption by cell incubation with methyl-␤-cyclodextrin (M␤CD) impaired specific HCN4 localization. It also shifted the midpoint of activation of the HCN4 current in HEK cells and of I f in SAN cells to the positive direction by 11.9 and 10.4 mV, respectively. These latter effects were not due to elevation of basal cyclic nucleotide levels because the cholesterol-depletion treatment did not alter the current response to cyclic nucleotides. In accordance with an increased I f , M␤CD-treated SAN cells showed large increases of diastolic depolarization slope (87%) and rate (58%). We also found that the kinetics of HCN4-and native f-channel deactivation were slower after lipid raft disorganization. In conclusion, our work indicates that pacemaker channels localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties. Key Words: HCN channels Ⅲ pacemaker current Ⅲ lipid rafts Ⅲ caveolin Ⅲ sinoatrial node T he sinoatrial node (SAN) is the region of the heart from which spontaneous action potentials originate and propagate to determine cardiac rhythm. This particular anatomical district is composed by specialized myocytes (pacemaker cells) whose activity is characterized by a slow diastolic depolarization phase at the end of the action potential. The pacemaker (I f ) current plays a key role in the generation of diastolic depolarization. f-Channels open toward the end of the action potential repolarization process and carry an inward current that depolarizes the membrane and drives the membrane potential up to threshold for initiating a new action potential. Although spontaneous activity is an intrinsic property of the heart, independent of innervation, fine modulation of heart rate is achieved through the release of the neurotransmitters norepinephrine (NE) and acetylcholine (ACh) by sympathetic and parasympathetic branches of the autonomic nervous system. It is well established that in cardiac cells NE and ACh, through specific ␤-adrenergic (␤-AR) and muscarinic (mAChR) receptors, modulate intracellular level of cAMP. Direct binding of cAMP to f-channels shifts their activation curve toward more depolarized potentials, thus increasing the steepness of the diastolic depolarization. 1 cAM...

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltagegated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin ؉ ), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin ؊ ) indicating that interaction with filamin A affects membrane localization. Also, in filamin ؊ cells the gating kinetics of HCN1 were strongly accelerated relative to filamin ؉ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.

The pacemaker current I f of the sinoatrial node (SAN) is a major determinant of cardiac diastolic depolarization and plays a key role in controlling heart rate and its modulation by neurotransmitters. Substantial expression of two different mRNAs (HCN4, HCN1) of the family of pacemaker channels (HCN) is found in rabbit SAN, suggesting that the native channels may be formed by different isoforms. Here we report the cloning and heterologous expression of HCN1 from rabbit SAN and its specific localization in pacemaker myocytes. rbHCN1 is an 822-amino acid protein that, in human embryonic kidney 293 cells, displayed electrophysiological properties similar to those of I f , suggesting that HCN1 can form a pacemaker channel. The presence of HCN1 in the SAN myocytes but not in nearby heart regions, and the electrophysiological properties of the channels formed by it, suggest that HCN1 plays a central and specific role in the formation of SAN pacemaker currents.

Agrin controls the formation of the neuromuscular junction. Whether it regulates the differentiation of other types of synapses remains unclear. Therefore, we have studied the role of agrin in cultured hippocampal neurons. Synaptogenesis was severely compromised when agrin expression or function was suppressed by antisense oligonucleotides and specific antibodies. The effects of antisense oligonucleotides were found to be highly specific because they were reversed by adding recombinant agrin and could not be detected in cultures from agrin-deficient animals. Interestingly, the few synapses formed in reduced agrin conditions displayed diminished vesicular turnover, despite a normal appearance at the EM level. Thus, our results demonstrate the necessity of agrin for synaptogenesis in hippocampal neurons.

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