Filamin A promotes the internalization of
HCN1 channels
Paul Feyen – 6323391
Supervisor & Assesor- Yoav Noam, Msc
Co-assesor- Wyste Wadman, Prof. Dr.
Abstract
Interactions with auxiliary proteins are a key molecular mechanism used by neurons to regulate ion channel function and trafficking. The aim of this study was to
delineate how the actin binding protein Filamin A (FLNa) affects the trafficking, distribution, and surface expression of hyperpolarization activated cyclic nucleotide
gated cation channel isoform 1 (HCN1) which underlie the generation of the Ih
current in the brain. To assess the regulatory effects of FLNa on HCN1, the study employed HCN1 constructs designed to interfere specifically with FLNa-HCN1
interaction. Transfection of HEK293 cells by these GFP-fused constructs was followed by confocal imaging. This revealed that interaction with FLNa induces intracellular clustering of HCN1 channels. Complimentary immunohistochemistry experiments further characterized this FLNa induced sequestration of HCN1 by showing that intracellular clusters mainly represent HCN1 localization in lysosomes. Next, we used a pharmacological manipulation to assess the potential mechanism underlying this effect. Results indicate that Filamin A regulates HCN1 channel surface expression by promoting its internalization in a dynamin dependent process. The data presented here describe FLNa as a negative regulator of HCN1 surface expression which promotes the channel’s internalization and endosomal processing.
Introduction
Electrical excitability and synaptic transmission is shaped in large part by the presence of specific ion channels with distinct functional properties that localize to discrete parts of a neuron (Lai and Jan, 2006). Accordingly, neuronal function can be influenced at different time scales and in a variety of ways by cellular mechanisms that mediate the function, expression, and trafficking of ion channels (Beck and Yaari, 2008, Lewis and Chetkovich, 2011). Two decades of research now points to the direct interaction of ion channels with accessory proteins as an important means of controlling the function and trafficking of ion channels (Vacher et al, 2008; Schwappag, 2008).
Here we aim to investigate the impact of channel-protein interaction on the trafficking and surface expression of HCN channels, which generate the hyperpolarization activated current, Ih. These voltage-gated channels encoded by the
HCN1-4 genes have diverse roles in the determination of neuronal excitability that include regulation of resting membrane potential and synaptic transmission (Robinson and Siegelbaum, 2003; Biel et al, 2009). The pore forming subunits assemble into HCN channel isoforms with distinct biophysical properties and distributions, thereby contributing to the heterogeneity of Ih across tissues and cell
types (Santoro et al, 2000; Moosmang et al, 2001). Isoform specific distributions of HCN channels are also evident at the sub-cellular level (Lörincz et al, 2002; Bender et al, 2007), and their sub-cellular localization within somatic, dendritic, and axonal compartments of specific neuron subtypes is developmentally regulated (Brewster et al, 2007; Bender et al, 2007). The specific patterns of subcellular expression critically influence the contribution that Ih makes to neuronal excitability (Santoro & Baram,
2003). For instance, whereas presynaptic localization modulates synaptic transmission rate (Huang et al, 2011), dendritic Ih has been proposed to normalize
temporal summation of synaptic input (Magee, 1999).
Interestingly, plasticity of HCN channel localization occurs not only in development, but has also been described in animal models of temporal lobe epilepsy, suggesting a potential role for aberrant channel trafficking and surface expression in neurological pathology (Shin et al, 2008; Jung et al, 2011). Despite its importance, the mechanisms underlying specific distributions of HCN channels are not yet fully understood, though these may be elucidated through the study of auxiliary proteins known to interact with HCN channels (Lewis et al, 2010).
Of particular interest is the regulation of HCN by the versatile cytoskeletal protein Filamin A (FLNa), which has been shown to interact directly and exclusively with the HCN1 channel isoform (Gravante, 2004). FLNa, whose developmental and subcellular expression patterns in the brain have been detailed (Sheen et al, 2002; Noam et al, 2012), is a key contributor to the mechanical and structural properties of the cytoskeleton through its binding of actin (Popowicz, 2006). Interestingly, FLNa also binds to a diverse set of membrane proteins, and has a role in regulating the surface expression and trafficking of several of these (Onoprishvili et al, 2003; Sverdlov et al, 2009; Minsaas et al, 2010), including certain ion channels (Petrecca et al, 2000; Kim
et al, 2007). The interaction of FLNa with HCN1 results from a 22 amino acid sequence that spans amino acid residues 694-715 at the C’ terminus of the channel (Gravante et al, 2004), and this sequence likely mediates FLNa’s regulation of HCN1. In the absence of FLNa, melanoma cells transfected with HCN1 show a continuous channel distribution along the membrane, whereas FLNa expressing melanoma cells showed HCN1 channel distribution in 'hotspots' along the membrane (Gravante et al, 2004). The knockout of FLNa not only influences channel surface expression but also has functional consequences; in the absence of FLNa Ih amplitude and current
density are enhanced, and channel gating is altered (Gravante et al, 2004). The cellular and molecular mechanisms underlying this regulatory effect of FLNa on HCN1 remain elusive.
Given the role of FLNa in the trafficking and surface expression of ion channels, we explored the hypothesis that the regulatory effects of FLNa on HCN1 are mediated by such cellular processes. To this end, this study employed a mutational approach that specifically targets HCN1-FLNa interaction mediated by the FLNa binding domain of HCN1. The resultant evidence from complimentary transfectional, immunocytochemical, and pharmacological experiments describe FLNa as a promoter of HCN1 channel internalization.
Experimental Procedures
Cell Cultures Condition
Human Embryo Kidney (HEK293) cells were obtained from ATCC, and were cultured in minimum essential medium (MEM) that was supplemented with 10% FCS, 2mM L-glutamine, 100 μg/ml penicillin, and 100 μg/ml streptomycin. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 and the medium was refreshed every 2-3 days. All culture reagents were obtained from Invitrogen.
cDNA Constructs
Three cDNA constructs were employed for this study, including two constructs of HCN1 and one of FLNa. The mouse HCN1 cDNA constructs were as follows; (1) A c-terminus GFP-conjugated construct (where GFP DNA was inserted between amino acid residues 885 and 886, see Noam et al. 2010), hereafter referred to as HCN1-GFP and (2) a similar c-terminus GFP-conjugated construct with a further deletion of the FLNa interaction domain (amino acid residues 694 to 715: SPPIQSPLATRTFHYASPTASQ), which is hereafter referred to as HCN1-Δ22-GFP. The two constructs were obtained from Dr. Chetkovich (Northwestern University) and Genscipt respectively and unpublished data from our lab confirms that both constructs yield a functional Ih in the heterologous systems employed in this study.
Human FLNa with N-terminal fusion of monomeric DsReD were obtained from Dr Nakamura (Harvard Medical School, MA, USA) and are hereafter referred to as DsRed-FLNa. DsRed-FLNa and endogeneous FLNa presented similar distributions in HEK293cells (see Figure 1).
Figure 1. From left to right: HEK 293 cells were tagged by mouse anti filamin and labeled by goat-anti-mouse alexa 488 or transfected with Dsred-FLNa. Endogeneous and exogeneous FLNa show the same distribution. Scale bar set to 5 μm.
Calcium Phosphate Transfection
Cells from culture were re-plated on 12mm coverslips in a 24 well dish and transfected two to three days later using the calcium phosphate transfection method. 500 µl of transfection solution was administered per well. Per well the solution contained 12.5mM CaCl2, 0.3 µg of each cDNA construct to be transfected,
and distilled water for a running total volume of 25 µl, which after 90 second incubation at 37.5 °C was diluted 1:1 with 2x BBS, before medium was added for a final solution volume of 500 µl. HEK293 cells were transfected either solely with one of the two HCN1 cDNA constructs, or co-transfected with Dsred-FLNa using a 1:1 DNA ratio.
Immunocytochemistry
Transfected cells were stained for endosomal markers and non-transfected cells for endogenous FLNa. Primary antibodies used for staining were monoclonal mouse anti-LAMP1 (1:200; Abcam; clone [H4A3]) for tagging lysosomes, mouse monoclonal anti-EEA1 (1:2000; BD Transduction Laboratories; clone 13) for tagging early endosomes, and mouse monoclonal anti-FLNa (1:1000; Millipore; clone PM6/317) for tagging endogenous FLNa. Secondary antibodies used for detection were goat-anti-mouse Alexa 488 (1:400; Invitrogen) or goat-goat-anti-mouse Alexa 635 (1:400; Invitrogen). Multiple protocols were applied for fixation and permeabilization to optimize signal quality; for staining of EEA1, endogenous FLNa, and endogenous actin, cells were fixed on ice in PBS-4%-PFA for 15 (EEA1) or 20 minutes. These cells, still on ice, were thoroughly washed with PBS, and subsequently permeabilized for 5 minutes using PBS-0.1%Triton-X. Cells tagged for lysosomes were fixed and permeabilized by 5 minute incubation in methanol at -20°C. Following 3 washes with PBS, cells were incubated in a PBS-5%BSA-1%NGS blocking solution for one hour, and then incubated for one hour or overnight with primary antibody in PBS-5%BSA. Following thorough washes with 1M PBS, cells were incubated for 30 minutes in secondary antibody solution prepared with PBS-5%NGS. After a final wash with PBS, the coverslips were mounted using Fluoromount-G (SouthernBiotech). All experiments were conducted 22-26 hours after transfection.
Figure 2. HEK293 cells co-transfected with DsRed-FLNa and HCN1-GFP (top row) show strong clustering of HCN1 channels whereas cell co-transfected with DsRed-FLNa and HCN1- Δ22-GFP present a more diffuse distribution. Scale bar set to 5 μm.
Dynamin Inhibition by Dynasore Administration
Dynamin dependent internalization was inhibited using 80 µM dynasore in DMSO (Sigma). Incubation began 24 hrs after co- transfection of HCN1-GFP and DsRed-FLNa. Cells were incubated for 2, 4, or 6 hrs, and control group was incubated for 6 hrs in DMSO. Cells were then fixed on ice in PBS-4%-PFA, thoroughly washed, and mounted as described above.
Results
FLNa binding domain mediates clustering of HCN1 channels
The inhibitory effect of FLNa on Ih amplitude and current density (Gravante et al,
2004) could result from a modification of the channel’s biophysical properties, or by a lower number of surface expressed channels. To investigate the possibility that the regulatory effects of FLNa on HCN1 are arbitrated by channel trafficking, HEK293 cells were co-transfected with HCN1-GFP or the mutant counterpart in which the FLNa interaction domain (Gravante et al, 2004) is deleted. Confocal imaging revealed that co-transfecting cells with HCN1-GFP and DsRed-FLNa yields a HCN channel distribution that is strikingly distinct from that of cells co-transfected with HCN1-Δ22-GFP and DsRed-FLNa (Figure 2). The fluorescence signal of HCN1-GFP channels was principally restricted to large distinct puncta, indicating a strong clustering of the channels. In contrast to this, the deletion construct was expressed in a more diffuse manner across the cell, and the distinct GFP positive puncta were absent. An observer blinded to experimental conditions was consistently able to identify the transfected HCN1 construct across multiple experiments on the basis of this distribution difference. These results indicate that interaction with FLNa leads to HCN1 channel clustering in putatively intracellular compartments.
Figure 3. HEK293cells co- transfected with HCN1-GFP and DsRed-FLNa. Top 2 Rows (L toR) showing HCN1-GFP; mouse-anti-LAMP1 tagged by goat-anti-mouse alexa635; merge of HCN1-GFP and LAMP1 signal; and DsredFLNa. HCN1-GFP shows a strong co-localization with LAMP1, suggesting HCN1 localization in lysosomes. Selection shown in middle row merged image is enlarged in bottom row. Scale bar set to 5 μm (top row) and 1 μm.
FLNa promotes the accumulation of HCN1 channels in lysosomal compartments
As FLNa has been reported to regulate the endosomal processing of several membrane proteins (Seck et al, 2003; Sverdlov et al, 2009; Minsaas et al, 2010), we hypothesized that the observed HCN1-GFP puncta reflect channel localization in endosomal organelles. To test this hypothesis, cells were immunostained in three separate experiments for early endosomal and lysosomal antigens following transfection with HCN1-GFP. These experiments revealed that the HCN1-GFP puncta show a partial colocalization with early endosomal marker (Figure 4). In contrast to this limited presence of HCN1 channels in early endosomes, HCN1-GFP puncta reliably showed a strong co-localization with the lysosomal marker, LAMP1 (Figure 3), indicating a preponderance of HCN1 channel localization in lysosomes. These results suggest that FLNa can mediate the presence of HCN1-GFP channels in endosomes involved in the internalization and degradation of membrane proteins. Interestingly, cells co-transfected with HCN1-GFP showed a reduced Ih amplitude
and current density compared to those co-transfected with HCN1-Δ22-GFP (data not shown), a finding that is in line with a role of FLNa in promoting the endocytosis and degradation of the channels, which would result in a net decrease of HCN1 surface expression.
Figure 4. HEK293 co- transfected with GFP and DsRed-FLNa Top Row (L toR) showing HCN1-GFP; mouse-anti-EEA1 tagged by goat-anti-mouse alexa635; merge of HCN1-GFP and EEA1 signal; and DsredFLNa. HCN1-GFP showed limited colocalization with early endosomal markers. Selection shown in top row merged image is enlarged in bottom row. Scale bar set to 5 μm (top row) and 1 μm.
Inhibition of dynamin dependent internalization leads to loss of FLNa mediated HCN1 clusters
The evidence presented thus far indicates a role for FLNa in mediating HCN1 surface expression; the interaction with FLNa leads to an intracellular accumulation of the channels in endocytic organelles and a reduction of Ih. In
order to investigate the possible mechanism by which FLNa promotes HCN1 channel internalization, we inhibited the function of dynamin, a GTPase which contributes vitally to many forms of endocytosis (Macia et al, 2006). Given that the HCN1-GFP puncta are primarily located in proteolytic lysosomal compartments, successful inhibition of the inward trafficking of the channel should result in a reduction of intracellular HCN1-GFP puncta. Indeed the inhibition of dynamin resulted in marked reduction of HCN1-GFP intracellular puncta, and the effect could be observed after only two hours of dynasore incubation (Figure 5). An observer blinded to experimental conditions was correctly able to distinguish dynasore treated cells from control cells across multiple experiments. Importantly, the distribution of HCN1-Δ22-GFP was not altered by dynasore administration (Figure 5). These results indicate that blocking dynasore dependent internalization leads to a reduction of FLNa mediated HCN1 clusters, and suggest that FLNa mediated clustering of HCN1 channels is dynamin dependent.
Figure 5. HEK293 co-transfected with DsRed-FLNa and HCN1-GFP (top row) or with DsRed-FLNa and HCN1- Δ22-GFP were incubated in 80μM dynasore or vehicle. Dynamin administration led to a reduction of HCN1-GFP clustering.
Discussion
By investigating the regulation of HCN1 channel distribution, trafficking, and surface expression, this study aimed to shed light on the cellular and molecular mechanisms that contribute to the versatile nature of Ih. The evidence
presented above of HEK293 cells transfected with either HCN1-GFP or its mutational counterpart HCN1-Δ22-GFP indicates that the interaction of the HCN1 isoform with FLNa has distinct regulatory consequences. Here we found that the interaction with FLNa induces clustering of HCN1 channels, and that these clusters represent HCN1 localization in endocytic organelles. Lastly, this study found that inhibition of dynamin mediated endocytosis causes a marked reduction of HCN1 clusters, suggesting that FLNa mediated clustering of HCN1 is a dynamin dependent process.
Prior to this study, knockout of FLNa had been shown to upregulate Ih, an effect
reflected in a decreased amplitude and current density of Ih (Gravante et al,
2004). The results of the present study put forward a likely underlying mechanism of this observation: if HCN1 can interact with FLNa, the channels are directed towards intracellular compartments, namely lysosomes. This internalization and subsequent degradation is likely to cause a decrease in the number of surface expressed channels, thereby leading to a reduced Ih current
density.
The internalization of HCN1 channels that is mediated by filamin depends on dynamin. We found that inhibiting the GTP hydrolyzing activity of dynamin by dynasore administration abolished HCN1-GFP clusters, which primarily reflect HCN1 localization in lysosomes. The exact role of dynamin could not be delineated by this study. Dynamin is involved in several forms of endocytosis including clathrin mediated endocytosis (Macia et al, 2006), a process known to regulate the surface expression of many membrane proteins expressed in the central nervous system including GABAa receptors (Kittler et al, 1999) and ionotropic glutamate receptors (Carroll et al, 1999). However dynamin also plays
a crucial role in non-clathrin mediated endocytosis, such as in the internalization of caveolae (Oh et al, 1998; Henley et al, 1998). Caveolae are membrane microdomains that along with other roles act to cluster membrane proteins and mediate their internalization (Parton and Simmons, 2007). Interestingly, FLNa can bind to the caveolae marker caveolin 1, and even promotes its clustering and internalization (Stahlhut and van Deurs, 2000; Sverdlov et al, 2009). Though the exact cascade of events cannot be determined with the data at hand, the internalization of HCN1 channels which is promoted by interaction with FLNa is a dynamin dependent process.
To investigate the basic mechanisms regulating HCN1 trafficking and surface expression this study employed HEK293 cells as a heterologous expression system and its translational value of must be carefully weighed. However, given that the channel and cytoskeletal protein have been co-immunoprecipitated from bovine brain (Gravante et al, 2004), it is likely that FLNa and HCN1 interact
in vivo. In the brain, FLNa might act to regulate channel localization. In dendrites
of pyramidal cells HCN1 density increases with distance from the soma where its surface expression can be 60 times lower than in distal dendrites (Lörincz et al, 2002). This somatodendritic gradient has been proposed to normalize temporal summation of synaptic input (Magee, 1999), modulate the integrative properties of neurons (Williams and Stuart, 2000), and to constrain synaptic plasticity (Nolan et al, 2004). Interestingly, FLNa expression is stronger in distal dendrites than in perisomatic regions of primary hippocampal neurons (Noam et al, under revision). Given that we demonstrated a role for FLNa in promoting the internalization of HCN1, this subcellular expression pattern of FLNa raises the possibility that FLNa contributes, in part, to the polarized subcellular distribution of HCN1 in dendrites of pyramidal neurons.
To conclude, HCN1 channel distribution, trafficking, and surface expression is modulated by the actin binding protein FLNa. The interaction of these proteins, mediated by a 22 amino acid sequence in HCN1, downregulates Ih by promoting
dynamin dependent internalization of the channel. Future studies should aim at evaluating the extent of FLNa's effects on Ih in neuronal cultures, organotypic
slices, or in vivo. What is also of interest is the identification of up and down stream modulators of FLNa's effect on HCN1, such as caveolae. The ability of HCN1 to carry out its physiological functions depends in large part on its sub-cellular localization; (1) Is it functionally integrated in the membrane? (2) In which part of the neuron is it located? Understanding the processes which determine the answers to these questions could pave the way to a better understanding of HCN1 physiology, and how this contributes to normal cognitive and behavioral functioning or to the pathogenesis of neurological disorders.
Acknowledgment
I thank Dr. Yoav Noam for his supervision and support during this study, Dr. Wytse Wadman for his advice, and Ronald Breedijk from the Center for Advanced
References
Beck H, Yaari Y (2008) Plasticity of intrinsic neuronal properties in CNS disorders. Nat. Rev. Neurosci. 9:357-369
Bender RA, Kirschstein T, Kretz O, Brewster AL, Richichi C, Ruschenschmidt C,
Shigemoto R, Beck H, Frotscher M, Baram TZ (2007) Localization of HCN1 channels to presynaptic compartments: novel plasticity that may contribute to hippocampal maturation. J. Neurosci. 27:4697-4706
Brewster AL, Chen Y, Bender RA, Yeh A, Shigemoto R, Baram TZ (2007) Quantitative analysis and subcellular distribution of mRNA and protein expression of the
hyperpolarization-activated cyclic nucleotide-gated channels throughout development in rat hippocampus. Cerb. Cortex. 17:702-712
Carroll R, Beattie E, Xia H, Luscher C, Altschuler Y, Nicoll R, Malenka R, von Zastrow R. Dynamin-dependent endocytosis of ionotropic glutamate receptors. PNAS. 96:285-290
Biel M, Wahl-Schott C, Michlakis S, Zong X (2009) Hyperpolarization-activated cation channels: from genes to function. Physiol. Rev. 89:847-885
Gravante B, Barbuti A, Milanesi R, Zappi I, Viscomi C, DiFrancesco D (2004)
Interaction of the pacemaker channel HCN1 with filamin A. J. Biol. Chem. 279:43847-43853
Henley, JR (1998) Dynamin-mediated internalization of caveolae. J. Cell Biol. 141:85– 99
Huang Z, Lujan R, Kaduring I, Uebele V, Renger J, Dolphin A, Sha M (2011) Presynaptic HCN1 channels regulated CaV3.2 activity and neurotransmission at select cortical synapses. Nat. Neurosci. 14:478-486
Jung S, Warner LN, Pitsch J, Becker AJ, Poolos NP (2011) Rapid loss of dendritic HCN channel expression in hippocampal pyramidal neurons following status epilepticus. J. Neurosci. 31:14291-14295
Kim EJ, Park JS, Um SJ (2007) Interacion with Filamin A stimulate surface expression of large-conductance Ca2+-activated K+ channels in the absence of direct actin binding. Mol. Pharmacol. 72:622-630
Kittler J, Delmas P, Jovanovic J, Brown D, Smart T, Moss C (1999) Constitutive endocytosis of GABAa receptors by an association with the adaptin AP2 complex modulates inhibitory synaptic currents in hippocampal neurons. J. Neurosci. 20:7972-7977
Lai HC, Jan LY (2006) The distribution and targeting of neuronal voltage-gated ion channels. Nat. Rev. Neurosci. 7:548-562
Lewis AS, Chetkovich DM (2011) HCN channels in behavior and neurological disease: Too hyper or not active enough? Mol. Cell. Neurosci. 46:357-367
Lewis AS, Estep CM, Chetkovich DM (2010) The fast and slow ups and downs of HCN channel regulation. Channels. 4:215-231
Lörincz A, Notomi T, Tamas G, Shigemoto R, Nusser Z (2002) Polarized and compartment dependent distribution of HCN1 in pyramidal cell dendrites. Nat. Neurosci. 5:1185-1193.
Macia, E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T (2006) Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10:839-850
Magee JC (1999) Dendritic Ih normalized temporal summation in hippocampal CA1 neurons. Nat. Neurosci. 2:848
Minsaas L, Planaguma J, Madziva M, Krakstad BF, Masia-Balague M, Katz AA, Aragay AM (2010) Filamin a binds to CCR2B and regulates its internalization. PLoS ONE. 17: 12212-12220
Moosmang S, Biel M, Hofmann F, Ludwig A (2001) Differential distribution of four hyperpolarization-activated cation channels in mouse brain. Biol. Chem. 380: 975– 980
Noam Y, Zha Q, Phan L, Wu RL, Chetkovich DM, Wadman WJ, Baram TZ (2010) Trafficking and surface expression of hyperpolarization-activated cyclic nucleotide-gated channels in hippocampal neurons. J. Biol. Chem. 285:14724-14736
Nolan M, Mallere G, Dudman J, Buhl D, Santoro B, Gibbs E, Vronskaya S,Buzaky G, Siegelbaum S, Kandel E, Morozov AA (2004) Behavioral Role for Dendritic
Integration: HCN1 Channels Constrain Spatial Memory and Plasticity at Inputs to Dendrites of CA1 Pyramidal Neurons. Cell. 119(5): 719-732
Oh P. et al (1998) Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTPdriven fission from the plasma membrane of endothelium. J. Cell Biol. 141: 101–114
Onoprishvili I, Andri Ml, Kramer HK, Ancevska-Taneva N, Hiller JM, Simon EJ (2003) Interaction between mu opioid receptor and filamin A is involved in receptor regulation and trafficking. Mol. Pharmacol. 64:1092-1100
Parton R, Simmons, K (2007) The multiple faces of caveolae. Nat. Rev. Mol. Cell Biol.8:185-194
Petrecca K, Miller DM, Shrier A (2000) Localization and enhanced current density of the Kv4.2 potassium channel by interaction with the actin binding protein filamin. J. Neurosci. 20: 8736-8744
Popowicz GM, Schleicher M, Noegel AA, Hoolak TA (2006) Filamins: Promiscuous organizers of the cytoskeleton. Trends Biochem. 31:411-419
Robinson RB, Siegelbaum Sa (2003) Hyperpolarization –activated cation currents: From molecules to physiological function. Annu. Rev. Physiol. 65:453-480
Santoro B, Chen S, Luthi A, Pavlidis P, Shumyatsky GP, Tibbs GR, Siegelbaum Sa (2000) Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. J. Neurosci. 20:5264-5275
Santoro B, Baram TZ (2003) The multiple personalities of h-channels. Trends Neurosci. 26:550-554
Schwappag, B (2008) An overview of trafficking and assembly of
neurotransmitter receptors and ion channels. Mol. Membr. Biol. 25:270-278 Sheen VL, Feng Y, Graham D, Takafuta T, Shapiro SS, Walsh Ca (2002) Filamin A and Filamin B are co-expressed within neurons during periods of neuronal migration and can physically interact. Hum Mol Gen 11:2845-2854
Shin M, Brager D, Jaramillo TC, Johnston D, Chetkovich DM (2008) Mislocalization of h channel subunits underlies h channelopathy in temporal lobe epilepsy. Neurobiol. Dis. 32:26-36
Stalhult M, van Deurs B (2000) Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton. Mol. Biol. Cell. 11:325-337
Sverdlov M, Shinin V, Place AT, Castellon M, Minshall RD (2009) Filamin A regulates caveolae internalization and trafficking in endothelial cells.
Vacher H, Mohapatra D, Trimmer J (2008) Localization and Targeting of Voltage-Dependent Ion Channels in Mammalian Central Neurons. Phys. Rev. 88:1407-1447 Williams Sr, Stuart GJ (2003) Voltage- and site-dependent control of the somatic impact of dendritic IPSPs. J. Neurosci. 23: 7358-7367
