Distribution of Ca2+ channels on cochlear outer hair cells revealed by fluorescent dihydropyridines

1996 ◽  
Vol 271 (3) ◽  
pp. C944-C949 ◽  
Author(s):  
T. Oshima ◽  
K. Ikeda ◽  
M. Furukawa ◽  
N. Ueda ◽  
H. Suzuki ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hair Cells ◽  
Laser Scanning ◽  
Basolateral Membrane ◽  
Outer Hair Cells ◽  
Spatial Integration ◽  
Heterogeneous Distribution ◽  
Voltage Dependent ◽  
Basolateral Plasma Membrane ◽  
Ca2 Channels

Physiological evidence has shown that cochlear outer hair cells (OHC) possess L-type voltage-dependent Ca2+ channels through which Ca2+ enters the OHC during depolarization. Their subcellular distribution has, however, remained unclear. In this study, the distribution of L-type Ca2+ channels on the basolateral plasma membrane of OHC has been demonstrated by the use of a laser scanning confocal microscope (LSCM) and a fluorescent probe DMBODIPY-DHP. The fluorescent staining pattern on the basolateral wall is nonuniform, suggesting a heterogeneous distribution of the channels in the plasma membrane. Direct imaging of intracellular Ca2+ visualized in real time by means of the LSCM and the fluorescent Ca2+ probe fluo 3 revealed temporal and spatial integration of Ca2+ movements and Ca2+ channel distribution. Exposure to high-K+ solution induced heterogeneity in the subcellular increase in the intracellular Ca2+ concentration. These results suggest that the heterogeneous distribution of L-type Ca2+ channels on the basolateral membrane might induce heterogeneous intracellular Ca2+ distribution during electrical activity in the OHC.

Two types of voltage-dependent potassium channels in outer hair cells from the guinea pig cochlea

1999 ◽  
Vol 277 (5) ◽  
pp. C913-C925 ◽  
Author(s):  
Thierry van den Abbeele ◽  
Jacques Teulon ◽  
Patrice Tran Ba Huy
Keyword(s):  
Guinea Pig ◽  
Hair Cells ◽  
Basolateral Membrane ◽  
Outer Hair Cells ◽  
Catalytic Subunit ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Guinea Pig Cochlea ◽  
Voltage Dependent ◽  
Inside Out

Cell-attached and cell-free configurations of the patch-clamp technique were used to investigate the conductive properties and regulation of the major K+channels in the basolateral membrane of outer hair cells freshly isolated from the guinea pig cochlea. There were two major voltage-dependent K+ channels. A Ca2+-activated K+ channel with a high conductance (220 pS, P K/ P Na= 8) was found in almost 20% of the patches. The inside-out activity of the channel was increased by depolarizations above 0 mV and increasing the intracellular Ca2+concentration. External ATP or adenosine did not alter the cell-attached activity of the channel. The open probability of the excised channel remained stable for several minutes without rundown and was not altered by the catalytic subunit of protein kinase A (PKA) applied internally. The most frequent K+ channel had a low conductance and a small outward rectification in symmetrical K+ conditions (10 pS for inward currents and 20 pS for outward currents, P K/ P Na= 28). It was found significantly more frequently in cell-attached and inside-out patches when the pipette contained 100 μM acetylcholine. It was not sensitive to internal Ca2+, was inhibited by 4-aminopyridine, was activated by depolarization above −30 mV, and exhibited a rundown after excision. It also had a slow inactivation on ensemble-averaged sweeps in response to depolarizing pulses. The cell-attached activity of the channel was increased when adenosine was superfused outside the pipette. This effect also occurred with permeant analogs of cAMP and internally applied catalytic subunit of PKA. Both channels could control the cell membrane voltage of outer hair cells.

scholarly journals Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles

1996 ◽  
Vol 316 (3) ◽  
pp. 999-1004 ◽  
Author(s):  
Lorella PASCOLO ◽  
Savino DEL VECCHIO ◽  
Ronald K. KOEHLER ◽  
J. Enrique BAYON ◽  
Cecile C. WEBSTER ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Experimental Conditions ◽  
Saturation Kinetics ◽  
Basolateral Plasma Membrane ◽  
Component C ◽  
Uptake Studies

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB–HSA affinity constants (K´f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 μM, K´f (litre/mol) was inversely related to [HSA], irrespective of the [Bt]/[HSA] ratio. K´f was 2.066×106+(3.258×108/[HSA]). When 50 mM KCl was iso-osmotically substituted for sucrose, the K´f value was significantly lower {2.077×106+(1.099×108/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] ⩽ 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with Km values of 28±7 and 57±8 nM respectively (mean±S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112±12 versus 45±4 pmol of UCB·mg-1 of protein·15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K´f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with Km values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K´f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

The CaV3.1 T-type Ca2+channel contributes to voltage-dependent calcium currents in rat outer hair cells

2008 ◽  
Vol 1201 ◽  
pp. 68-77 ◽  
Author(s):  
Akira Inagaki ◽  
Shinya Ugawa ◽  
Hisao Yamamura ◽  
Shingo Murakami ◽  
Shoichi Shimada
Keyword(s):  
Hair Cells ◽  
Outer Hair Cells ◽  
Calcium Currents ◽  
Voltage Dependent

scholarly journals Changes in plasma membrane structure and electromotile properties in prestin deficient outer hair cells

Cytoskeleton ◽  
2009 ◽  
pp. n/a-n/a ◽  
Author(s):  
David Z.Z. He ◽  
Shuping Jia ◽  
Takashi Sato ◽  
Jian Zuo ◽  
Leonardo R. Andrade ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hair Cells ◽  
Membrane Structure ◽  
Outer Hair Cells

Immunocytochemical studies of the Na-K-Cl cotransporter of shark kidney

1996 ◽  
Vol 270 (6) ◽  
pp. F927-F936 ◽  
Author(s):  
D. Biemesderfer ◽  
J. A. Payne ◽  
C. Y. Lytle ◽  
B. Forbush
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Basolateral Membrane ◽  
Distal Tubule ◽  
Rectal Gland ◽  
Apical Plasma Membrane ◽  
Secretory Cells ◽  
Weak Staining ◽  
Basolateral Plasma Membrane ◽  
Kidney Functions

The Na-K-Cl cotransporter (NKCC or BSC) has been described in numerous secretory and reabsorptive epithelia and is an important part of the mechanism of NaCl reabsorption in both the mammalian and elasmobranch kidneys. We have recently developed a panel of four monoclonal antibodies (MAbs) raised to the 195-kDa Na-K-Cl cotransport protein of the shark rectal gland (sNKCC1), which is expressed along the basolateral plasma membrane of secretory cells in this tissue (29). Here, we report immunologic studies of the Na-K-Cl cotransporter in the kidney of the dogfish shark Squalus acanthias. Western blot analysis of shark renal microsomes using MAbs J3, J7, and J25 identified proteins of approximately 195 and 150 kDa, whereas MAb J4 was not reactive. To define the cellular and subcellular distribution of the cotransport protein, immunofluorescence and immunoelectron microscopy studies were performed on fixed kidneys. Immunofluorescence microscopy on semithin (0.5-micron) cryosections demonstrated that MAbs J3, J7, and J25 intensely stained the apical plasma membrane of all distal tubule segments. Weak staining was also seen along the basolateral membrane of most distal nephrons. Immunoelectron microscopy confirmed this observation and showed that some of these segments were morphologically similar to diluting segments from other species. MAbs also reacted with the brush border and, to a lesser extent, the basolateral membrane of proximal tubules. This study supports the hypothesis that the lateral bundle zone of the elasmobranch kidney functions as a countercurrent exchanger and is consistent with the presence of multiple isoforms of the Na-K-Cl cotransporter in the shark kidney.

scholarly journals Calcium efflux and cycling across the synaptosomal plasma membrane

1985 ◽  
Vol 226 (1) ◽  
pp. 225-231 ◽  
Author(s):  
R Snelling ◽  
D Nicholls
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Potential Gradient ◽  
Electrochemical Potential ◽  
Electrochemical Gradient ◽  
Calcium Efflux ◽  
Normal Medium ◽  
Voltage Dependent ◽  
Synaptosomal Plasma Membrane ◽  
Ca2 Channels

Ca2+ efflux from intact synaptosomes is investigated. Net efflux can be induced by returning synaptosomes from media with elevated Ca2+ or high pH to a normal medium. Net Ca2+ efflux is accelerated when the Na+ electrochemical potential gradient is collapsed by veratridine plus ouabain. Under steady-state conditions at 30 degrees C, Ca2+ cycles across the plasma membrane at 0.38 nmol . min-1 . mg-1 of protein. Exchange is increased by 145% by veratridine plus ouabain, both influx and efflux being increased. Increased influx is probably due to activation of voltage-dependent Ca2+ channels, since it is abolished by verapamil. The results indicate that, at least under conditions of low Na+ electrochemical gradient, some pathway other than a Na+/Ca2+ exchange must operate in the plasma membrane to expel Ca2+.

Dynamic tether force measurements of the outer hair cells plasma membrane with optical tweezers

2001 ◽  
Author(s):  
Bahman Anvari ◽  
Zhiwei Li ◽  
Masayoshi Takashima ◽  
Peter Brecht ◽  
Jorge H. Torres ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Optical Tweezers ◽  
Hair Cells ◽  
Outer Hair Cells ◽  
Force Measurements

Voltage-dependent channels in dissociated outer hair cells of the guinea pig

1994 ◽  
Vol 251 (S1) ◽  
pp. S57-S60 ◽  
Author(s):  
T. Nakagawa ◽  
S. Kakehata ◽  
N. Akaike ◽  
S. Komune ◽  
T. Takasaka ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Hair Cells ◽  
Outer Hair Cells ◽  
Voltage Dependent

Taurocholate transport by basolateral plasma membrane vesicles isolated from developing rat liver

1985 ◽  
Vol 248 (6) ◽  
pp. G648-G654
Author(s):  
F. J. Suchy ◽  
S. M. Courchene ◽  
B. L. Blitzer
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Plasma Membrane Vesicles ◽  
Adult Rat ◽  
Basolateral Plasma Membrane ◽  
Taurocholate Transport

Taurocholate transport was characterized in basolateral plasma membrane vesicles prepared from the livers of 14-day-old Sprague-Dawley rats using a self-generating Percoll gradient method. Liver plasma membrane protein yield, intravesicular volume, and enrichments of various marker enzymes were similar to those obtained for vesicles from adult rat liver. The basolateral marker enzyme Na+-K+-ATPase was enriched 26-fold in the suckling rat basolateral membrane fraction while the bile canalicular marker enzymes alkaline phosphatase and Mg2+-ATPase were enriched only 3- and 5-fold, respectively. The activities of marker enzymes for endoplasmic reticulum, mitochondria, or lysosomes were not enriched compared with homogenate. In the presence of an inwardly directed 100 mM Na+ gradient, vesicle accumulation of taurocholate transiently reached a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot") in suckling and adult membrane vesicles, but the initial rate of taurocholate entry and peak intravesicular accumulation were markedly decreased in suckling compared with adult membrane vesicles. In the presence of an inwardly directed 100 mM K+ gradient, the rate of uptake was slower, and no overshoot occurred in either suckling or adult rat vesicles. The decreased rate of Na+-coupled taurocholate uptake by membrane vesicles from suckling rat liver could not be explained on the basis of more rapid dissipation of the transmembrane Na+ gradient. Kinetic studies demonstrated saturable, Na+-dependent taurocholate uptake for both suckling and adult vesicles. However, the Vmax for taurocholate uptake in suckling rat vesicles was less than half of the adult rate (2.46 +/- 0.13 vs. 5.25 +/- 0.22 nmol X mg prot-1 X min-1, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation, Chemical Characterization, and Immunohistochemical Localization of a Protein from the Basolateral Plasma Membrane of the Rat Intestinal Absorptive Cell

1989 ◽  
Vol 44 (3-4) ◽  
pp. 289-295
Author(s):  
H. Schiechl
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Basolateral Membrane ◽  
Chemical Characterization ◽  
Performic Acid ◽  
Major Protein ◽  
Absorptive Cell ◽  
Isolation And Purification ◽  
Basolateral Plasma Membrane ◽  
Small Intestinal

Abstract Rat, Intestinal Absorptive Cell, Basolateral Plasma Membrane, Protein Isolation. Chemical Characterization The protein pattern of the basolateral membrane (BLM) of the rat small intestinal absorptive cell shows about 20 major and a multitude of minor bands. A simple and efficient method is described for isolation and purification of a major protein in the 17 kDa molecular weight (M W)-range called Prot 17 . The isolated BLM of intestinal epithelial cells was dissolved in buffer 1 (Tris/HCl, 2% SD S, 10% glycerol, 5% β-m ercaptoethanol. pH 6.8) and subsequently dialyzed for 4 h against buffer 2 (Tris/glycine, pH 8.3) and then for 12 h against buffer 2 containing 25% methanol. The resulting precipitate contained Prot 17 and phospholipids in the form of liposom es. A ll other BLM proteins remained dissolved in the supernatant. Chemical characterization of Prot 17 suggested that it is an integral membrane protein amounting to about 5% of the total BLM protein. Amino acid analysis revealed a MW of 17.6 kDa. The Prot 17 molecule did not contain any PAS-positive carbohydrates. In its isolated form, and apparently also in the BLM . Prot 17 occurred as a polymerized structure with a MW of about 90 kD a. By dissolution in buffer 1 and heating to 100 °C for 1 min the complex was split into its 17 kD a subunits. By oxidation with performic acid it was also broken down into its subunits. A specific antiserum against Prot 17 was obtained from immunized Balb/c mice. Immunofluorescence labelling of rat small intestinal sec­tions with this serum showed that Prot 17 was not a BLM -specific protein. It occurred in both plasma membrane domains of the intestinal absorptive cell.

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