Renal Epithelial Cells Show Nonselective Cation Channel Activity and Express a Gene Related to the cGMP-Gated Photoreceptor Channel

1993 ◽  
pp. 147-164
Author(s):  
Christoph Korbmacher ◽  
Colin J. Barnstable
Keyword(s):  
Epithelial Cells ◽  
Cation Channel ◽  
Nonselective Cation Channel ◽  
Channel Activity ◽  
Renal Epithelial Cells

scholarly journals Characterization of Na+-permeable Cation Channels in LLC-PK1 Renal Epithelial Cells

2004 ◽  
Vol 279 (19) ◽  
pp. 20137-20146 ◽  
Author(s):  
Malay K. Raychowdhury ◽  
Cristina Ibarra ◽  
Alicia Damiano ◽  
George R. Jackson ◽  
Peter R. Smith ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Channel ◽  
Tubular Epithelial Cell ◽  
Cation Channel ◽  
Cation Channels ◽  
Epithelial Cell Line ◽  
Selectivity Ratio ◽  
Channel Activity ◽  
Renal Epithelial Cells ◽  
Reverse Transcription Pcr

In this study, the presence of Na+-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patch-clamp analysis under cell-attached conditions indicated the presence of spontaneously active Na+-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an ∼3:1 Na+/K+permeability-selectivity ratio. The Na+-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na+channel activity. Spontaneous and vasopressin-induced Na+channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the αENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although αENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on αENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na+-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na+transport mechanisms in the mammalian proximal nephron.

Cyclic GMP-activated channel activity in renal epithelial cells (A6)

1991 ◽  
Vol 1070 (1) ◽  
pp. 152-156 ◽  
Author(s):  
Yoshinori Marunaka ◽  
Akito Ohara ◽  
Paul Matsumoto ◽  
Douglas C. Eaton
Keyword(s):  
Epithelial Cells ◽  
Cyclic Gmp ◽  
Channel Activity ◽  
Renal Epithelial Cells

Ca(2+)-activated nonselective cation channel in apical membrane of vestibular dark cells

1992 ◽  
Vol 262 (6) ◽  
pp. C1423-C1429 ◽  
Author(s):  
D. C. Marcus ◽  
S. Takeuchi ◽  
P. Wangemann
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Cation Channel ◽  
Nonselective Cation Channel ◽  
Channel Activity ◽  
Dark Cells ◽  
Linear Current ◽  
Current Voltage ◽  
K Secretion ◽  
Vestibular Dark Cells

Patch-clamp recordings were made on cell-attached and excised apical membrane from dark cells of the semicircular canal of the gerbil. These cells are thought to secrete K+ and absorb Na+ from the luminal fluid (endolymph). Single-channel events were identified as being equally conductive (27.6 +/- 0.4 pS; n = 48) for K+, Na+, Rb+, Li+, and Cs+ and 1.4 times more permeable to NH4+ but not permeable to Cl-, Ca2+, Ba2+, nor to N-methyl-D-glucamine. The channels displayed linear current-voltage relations that passed nearly through the origin (intercept: -2.6 +/- 0.5 mV; n = 48) when conductive monovalent cations were present on both sides of the membrane in equal concentrations. Channel activity required the presence of Ca2+ at the cytosolic face; there was no activity at less than or equal to 10(-7) M Ca2+ and full activity at greater than or equal to 10(-5) M Ca2+. Cell-attached recordings had a mean reversal voltage of -36.4 +/- 7.9 mV (n = 7), which was interpreted to reflect the intracellular potential of dark cells under the present conditions. We have identified a nonselective cation channel in the apical membrane of vestibular dark cells that might participate in K+ secretion or Na+ absorption under stimulated conditions, but the density appears to be insufficient to fully account for the transepithelial K+ flux.

scholarly journals Loss of primary cilia increases polycystin-2 and TRPV4 and the appearance of a nonselective cation channel in the mouse cortical collecting duct

2019 ◽  
Vol 317 (3) ◽  
pp. F632-F637 ◽  
Author(s):  
Takamitsu Saigusa ◽  
Qiang Yue ◽  
Marlene A. Bunni ◽  
P. Darwin Bell ◽  
Douglas C. Eaton
Keyword(s):  
Collecting Duct ◽  
Cation Channel ◽  
Conditional Knockout ◽  
Nonselective Cation Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Duct Cells ◽  
Collecting Ducts ◽  
Collecting Duct Cells

Flow-related bending of cilia results in Ca2+ influx through a polycystin-1 (Pkd1) and polycystin-2 (Pkd2) complex, both of which are members of the transient receptor potential (TRP) family (TRPP1 and TRPP2, respectively). Deletion of this complex as well as cilia result in polycystic kidney disease. The Ca2+ influx pathway has been previously characterized in immortalized collecting duct cells without cilia and found to be a 23-pS channel that was a multimere of TRPP2 and TRPV4. The purpose of the present study was to determine if this TRPP2 and TRPV4 multimere exists in vivo. Apical channel activity was measured using the patch-clamp technique from isolated split-open cortical collecting ducts from adult conditional knockout mice with ( Ift88flox/flox) or without ( Ift88−/−) cilia. Single tubules were isolated for measurements of mRNA for Pkd1, Pkd2, Trpv4, and epithelial Na+ channel subunits. The predominant channel activity from Ift88flox/flox mice was from epithelial Na+ channel [5-pS Na+-selective channels with long mean open times (475.7 ± 83.26 ms) and open probability > 0.2]. With the loss of cilia, the predominant conductance was a 23-pS nonselective cation channel (reversal potential near 0) with a short mean open time (72 ± 17 ms), open probability < 0.08, and a characteristic flickery opening. Loss of cilia increased mRNA levels for Pkd2 and Trpv4 from single isolated cortical collecting ducts. In conclusion, 23-pS channels exist in vivo, and activity of this channel is elevated with loss of cilia, consistent with previous finding of an elevated-unregulated Ca2+-permeable pathway at the apical membrane of collecting duct cells that lack cilia.

A mechanogated nonselective cation channel in proximal tubule that is ATP sensitive

2002 ◽  
Vol 283 (1) ◽  
pp. F93-F104 ◽  
Author(s):  
Craig G. Hurwitz ◽  
Vivian Y. Hu ◽  
Alan S. Segal
Keyword(s):  
Proximal Tubule ◽  
Basolateral Membrane ◽  
Cation Channel ◽  
Transepithelial Transport ◽  
Single Channels ◽  
Epithelial Polarity ◽  
Nonselective Cation Channel ◽  
Channel Activity ◽  
Entry Pathway ◽  
Hypotonic Swelling

Ion channels that are gated in response to membrane deformation or “stretch” are empirically designated stretch-activated channels. Here we describe a stretch-activated nonselective cation channel in the basolateral membrane (BLM) of the proximal tubule (PT) that is nucleotide sensitive. Single channels were studied in cell-intact and cell-free patches from the BLM of PT cells that maintain their epithelial polarity. The limiting inward Cs+ conductance is ∼28 pS, and channel activity persists after excision into a Ca2+- and ATP-free bath. The stretch-dose response is sigmoidal, with half-maximal activation of about −19 mmHg at −40 mV, and the channel is activated by depolarization. The inward conductance sequence is: NH[Formula: see text] ∼ Cs+ ∼ Rb+> K+ ∼ Na+ ∼ Li+ > Ca2+ ∼ Ba2+> N-methyl-d-glucamine ∼ tetraethylammonium. The venom of the common Chilean tarantula, Grammostola spatulata, completely blocks channel activity in cell-attached patches. Hypotonic swelling reversibly activates the channel. Intracellular ATP concentration ([ATP]i) reversibly blocks the channel (inhibitory constant ∼0.48 mM), suggesting that channel function is coupled to the metabolic state of the cell. We conclude that this channel may function as a Ca2+ entry pathway and/or be involved in regulation of cell volume. We speculate this channel may be important when [ATP]i is depleted, as occurs during periods of increased transepithelial transport or with ischemic injury.

scholarly journals Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells

2009 ◽  
Vol 296 (1) ◽  
pp. F87-F97 ◽  
Author(s):  
Malay K. Raychowdhury ◽  
Arnolt J. Ramos ◽  
Peng Zhang ◽  
Margaret McLaughin ◽  
Xiao-Qing Dai ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Signaling Pathway ◽  
Primary Cilium ◽  
Primary Cilia ◽  
Sensory Function ◽  
Vasopressin Receptor ◽  
Channel Activity ◽  
Renal Epithelial Cells ◽  
Selective Channel ◽  
Cation Selective Channel

The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK1 renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R- gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK1 cells stained with anti-V2R antibodies and in LLC-PK1 cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 μM AVP ( trans) or forskolin ( cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.

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