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 ...
The anion conductance in primary cultures of rat inner medullary collecting duct cells was studied using perforated-patch whole-cell clamp technique. Depolarizations above 0 mv induced an outward anionic current with a time-dependent activation (Iovt) exhibiting a similar conductivity to Cl- and HCO3-. Iovt showed half-maximal activation around 32 mV with a slope factor of 23 mV, and showed a voltage-dependent activation time course that was well fitted by a sum of two exponential functions. Iovt was potentiated when external pH or external Ca2+ was increased and was blocked by external DIDS, DPC and furosemide. These characteristics of Iovt resemble that of the ClC-K1 channels mediated currents; however, anion substitution studies showed that Iovt exhibits a Br->Cl->I->NO3- conductivity sequence, different from that observed in the ClC-K1 channels-mediated conductance. We suggest that, in inner medullary collecting duct cells, ClC-K channels of an unidentified type give rise to this Cl- and HCO3- conductance. This is the first study of a channel-mediated HCO3- current in kidney tubular cells.
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