Endogenous channels in HEK cells and potential roles in HCN ionic current measurements

Varghese, Anthony; TenBroek, Erica M.; Coles, James A.; Sigg, Daniel C.

doi:10.1016/j.pbiomolbio.2005.05.002

Cited by 52 publications

(39 citation statements)

References 36 publications

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“…In a general way, this result agrees with previous observations of HCN-RNA in kidney cells (44,57). We also show that IMCD cells express antigens compatible with the presence of HCN2 channel proteins at the basolateral membrane.…”

Section: Discussionsupporting

confidence: 93%

“…Although I h and HCN gene expression is observed, almost exclusively, in excitable cells, a Northern blot study performed in mouse has shown the expression in liver and kidney of a splice variant of the HCN3 mRNA expressed in brain (44) while, more recently, the presence of mRNA encoding HCN2 and HCN3 in the kidney-derived transformed HEK293 cell was reported (57). If nonexcitable cells may express HCN genes, then the presence of the corresponding I h current and HCN channels in such cells is a possibility deserving careful attention.…”

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confidence: 99%

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A hyperpolarization-activated, cyclic nucleotide-gated, (I_h-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells

Bolívar¹,

Tapia²,

Arenas³

et al. 2008

American Journal of Physiology-Cell Physiology

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A hyperpolarization-activated, cyclic nucleotide-gated, (Ihlike) cationic current and HCN gene expression in renal inner medullary collecting duct cells. Am J Physiol Cell Physiol 294: C893-C906, 2008. First published January 16, 2008 doi:10.1152/ajpcell.00616.2006The cation conductancein primary cultures of rat renal inner medullary collecting duct was studied using perforated-patch and conventional whole cell clamp techniques. Hyperpolarizations beyond Ϫ60 mV induced a time-dependent inward nonselective cationic current (Ivti) that resembles the well-known hyperpolarization-activated, cyclic nucleotidegated Ih and If currents. Ivti showed a half-maximal activation around Ϫ102 mV with a slope factor of 25 mV. It had a higher conductance (but, at its reversal potential, not a higher permeability) for K ϩ than for Na ϩ (gK ϩ /gNa ϩ ϭ 1.5), was modulated by cAMP and blocked by external Cd 2ϩ (but not Cs ϩ or ZD-7288), and potentiated by a high extracellular K ϩ concentration. We explored the expression of the Ih channel genes (HCN1 to -4) by RT-PCR. The presence of transcripts corresponding to the HCN1, -2, and -4 genes was observed in both the cultured cells and kidney inner medulla. Western blot analysis with HCN2 antibody showed labeling of ϳ90-and ϳ120-kDa proteins in samples from inner medulla and cultured cells. Immunocytochemical analysis of cell cultures and inner medulla showed the presence of HCN immunoreactivity partially colocalized with the Na ϩ -K ϩ -ATPase at the basolateral membrane of collecting duct cells. This is the first evidence of an Ih-like cationic current and HCN immunoreactivity in either kidney or any other nonexcitable mammalian cells. kidney; hyperpolarization-activated current; nonselective cation channel; sodium transport HYPERPOLARIZATION-ACTIVATED, cyclic nucleotide-gated, cationic nonselective (HCN) currents termed I h , I f , I q , or inward pacemaker currents are found in a wide variety of excitable cells (1,21,32,41). The I h channels activate, with a slow time course, at hyperpolarizing voltages beyond Ϫ60 mV (near the resting potential of most cells) and are permeable to both Na ϩ and K ϩ . They are regulated by cyclic nucleotides (cAMP and cGMP) and external K ϩ and blocked by external Cs ϩ . The genes coding for I h channels form the HCN family, with four members (HCN1 to -4) in mammals. Each HCN isoform is able to form homomeric conducting channels that differ in their kinetics, steady-state voltage dependence, and sensitivity to modulation by cAMP (1,21,41,45). HCN isoforms may also coassemble to form heteromeric channel complexes (54, 27).Although I h and HCN gene expression is observed, almost exclusively, in excitable cells, a Northern blot study performed in mouse has shown the expression in liver and kidney of a splice variant of the HCN3 mRNA expressed in brain (44) while, more recently, the presence of mRNA encoding HCN2 and HCN3 in the kidney-derived transformed HEK293 cell was reported (57). If nonexcitable cells may express HCN genes, then the presence of the ...

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Section: Discussionsupporting

confidence: 93%

mentioning

confidence: 99%

A hyperpolarization-activated, cyclic nucleotide-gated, (I_h-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells

Bolívar¹,

Tapia²,

Arenas³

et al. 2008

American Journal of Physiology-Cell Physiology

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“…HEK-293 cells are an established model for studying heterologously expressed ion channels (Varghese et al, 2006), and most data on bestrophin-1 ion channel function have been obtained with measurements in this cell line Pusch, 2004). To increase cytosolic free Ca 2+ , the cells were additionally transfected with the human P2Y2 receptor and stimulated with ATP.…”

Section: Discussionmentioning

confidence: 99%

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Milenkovic

Röhrl

Weber

et al. 2011

Journal of Cell Science

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SummaryBestrophin-1, an integral membrane protein encoded by the BEST1 gene, is localized predominantly to the basolateral membrane of the retinal pigment epithelium. Mutations in the BEST1 gene have been associated with Best vitelliforme macular dystrophy (BMD), a central retinopathy with autosomal dominant inheritance and variable penetrance. Over 120 disease-causing mutations are known, the majority of which result in amino acid substitutions within four mutational hot-spot regions in the highly conserved N-terminal half of the protein. Although initially thought to impair Cl -channel function, the molecular pathology of BEST1 mutations is still controversial. We have analyzed the subcellular localization of 13 disease-associated BEST1 mutant proteins in polarized MDCK II cells, an established model of apical to basolateral protein sorting. Immunostaining demonstrated that nine of the 13 mutant proteins failed to integrate into the cell membrane. The defective proteins were predominantly retained in the cytoplasm, whereas wild-type bestrophin-1 revealed cell membrane localization. Functional analysis of I -fluxes in HEK-293 cells showed that all mutants exhibited a significant reduction in anion conductance. Our data indicate that defective intracellular trafficking could be a common cause of BMD accompanied by impaired anion conductance, representing a loss of anion channel function that is probably due to mistargeting of mutant protein.

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“…The stimulated APs displayed a large variation in their phenotypes, reflecting the randomness of the amount of cDNA taken up by a cell via routine transfection. At room temperature, the maximum diastolic potentials were Ϫ78Ϯ7 mV with an APD at 90% repolarization (APD 90 ) value of 475Ϯ33 ms (nϭ5). Our own observations and data from others 89,90 suggested that HEK293 cells express endogenous voltage-gated outward currents.…”

Section: Conversion Of Nonexcitable Cells To Self-contained Biologicamentioning

confidence: 99%

“…At room temperature, the maximum diastolic potentials were Ϫ78Ϯ7 mV with an APD at 90% repolarization (APD 90 ) value of 475Ϯ33 ms (nϭ5). Our own observations and data from others 89,90 suggested that HEK293 cells express endogenous voltage-gated outward currents. This allowed us to omit HERG, and include HCN1 channel in our assay to test whether additional expression of I HCN1 would suffice to generate spontaneously oscillating APs in HEK293 cells.…”

Section: Conversion Of Nonexcitable Cells To Self-contained Biologicamentioning

confidence: 99%

Biological Therapies for Cardiac Arrhythmias

Cho¹,

Marbán²

2010

Circulation Research

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Endogenous channels in HEK cells and potential roles in HCN ionic current measurements

Cited by 52 publications

References 36 publications

A hyperpolarization-activated, cyclic nucleotide-gated, (I_h-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells

A hyperpolarization-activated, cyclic nucleotide-gated, (I_h-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Biological Therapies for Cardiac Arrhythmias

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Endogenous channels in HEK cells and potential roles in HCN ionic current measurements

Cited by 52 publications

References 36 publications

A hyperpolarization-activated, cyclic nucleotide-gated, (Ih-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells

A hyperpolarization-activated, cyclic nucleotide-gated, (Ih-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Biological Therapies for Cardiac Arrhythmias

Contact Info

Product

Resources

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A hyperpolarization-activated, cyclic nucleotide-gated, (I_h-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells

A hyperpolarization-activated, cyclic nucleotide-gated, (I_h-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells