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Activation of Na+-permeant cation channel by stretch and cyclic AMP-dependent phosphorylation in renal epithelial A6 cells.

Marunaka Y, Shintani Y, Downey GP, Niisato N - J. Gen. Physiol. (1997)

Bottom Line: These observations led us to the conclusion that a kinetic model "CL <--> CS <--> O" was the most suitable among three possible linear models.According to this model, IBMX or stretch would decrease the leaving rate of the channel for CL from CS, resulting in an increase in Po.We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cellular and Molecular Physiology, Division of Respiratory Research, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada M5G 1X8. marunaka@sickkids.on.ca

ABSTRACT
It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na+ absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na+, K+, Li+, and Cs+, but little selectivity for Ca2+ (PCa/PNa < 0.005) or Cl- (PCl/PNa < 0.01), identifying it as an NSC channel. Stretch had no additional effects on the open probability (Po) of the IBMX-activated channel. This channel had one open ("O") and two closed (short "CS" and long "CL") states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the Po of the channel without any detectable changes in the mean open or closed times. These observations led us to the conclusion that a kinetic model "CL <--> CS <--> O" was the most suitable among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of the channel for CL from CS, resulting in an increase in Po. Cytochalasin D pretreatment abolished the response to stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.

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The open  probability (Po) of the  IBMX (1 mM)-activated  channel. (A) A typical  time course of IBMX-induced change in Po  obtained from a cell- attached patch. Similar  time courses could be  obtained in three more  individual cell-attached  patches. (B) Effects of  IBMX of different concentrations on Po. The  pipette potential was 0  mV. n = 4–11.
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Figure 2: The open probability (Po) of the IBMX (1 mM)-activated channel. (A) A typical time course of IBMX-induced change in Po obtained from a cell- attached patch. Similar time courses could be obtained in three more individual cell-attached patches. (B) Effects of IBMX of different concentrations on Po. The pipette potential was 0 mV. n = 4–11.

Mentions: Fig. 1 illustrates representative single-channel current traces from a cell-attached patch formed on the apical membrane at the resting membrane potentials before and after application of 1 mM IBMX. Before application of IBMX, the Po of the channel was low (Fig. 1 A). IBMX stably increased the Po for up to 30 min after its application (Fig. 1 B). The time course of IBMX-induced increase in Po of the channel is shown in Fig. 2 A. The Po reached a stable value ∼3–4 min after application of 1 mM IBMX and remained elevated throughout the time period of our measurement (at least 30 min). The effects of IBMX on Po increased in a dose-dependent manner (Fig. 2 B). IBMX significantly increased intracellular cAMP concentrations from 230 ± 20 fmol/105 cells (basal) to 540 ± 40 at 3 min and 1,180 ± 220 fmol/ 105 cells at 30 min after addition of IBMX (1 mM; n = 4).


Activation of Na+-permeant cation channel by stretch and cyclic AMP-dependent phosphorylation in renal epithelial A6 cells.

Marunaka Y, Shintani Y, Downey GP, Niisato N - J. Gen. Physiol. (1997)

The open  probability (Po) of the  IBMX (1 mM)-activated  channel. (A) A typical  time course of IBMX-induced change in Po  obtained from a cell- attached patch. Similar  time courses could be  obtained in three more  individual cell-attached  patches. (B) Effects of  IBMX of different concentrations on Po. The  pipette potential was 0  mV. n = 4–11.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229362&req=5

Figure 2: The open probability (Po) of the IBMX (1 mM)-activated channel. (A) A typical time course of IBMX-induced change in Po obtained from a cell- attached patch. Similar time courses could be obtained in three more individual cell-attached patches. (B) Effects of IBMX of different concentrations on Po. The pipette potential was 0 mV. n = 4–11.
Mentions: Fig. 1 illustrates representative single-channel current traces from a cell-attached patch formed on the apical membrane at the resting membrane potentials before and after application of 1 mM IBMX. Before application of IBMX, the Po of the channel was low (Fig. 1 A). IBMX stably increased the Po for up to 30 min after its application (Fig. 1 B). The time course of IBMX-induced increase in Po of the channel is shown in Fig. 2 A. The Po reached a stable value ∼3–4 min after application of 1 mM IBMX and remained elevated throughout the time period of our measurement (at least 30 min). The effects of IBMX on Po increased in a dose-dependent manner (Fig. 2 B). IBMX significantly increased intracellular cAMP concentrations from 230 ± 20 fmol/105 cells (basal) to 540 ± 40 at 3 min and 1,180 ± 220 fmol/ 105 cells at 30 min after addition of IBMX (1 mM; n = 4).

Bottom Line: These observations led us to the conclusion that a kinetic model "CL <--> CS <--> O" was the most suitable among three possible linear models.According to this model, IBMX or stretch would decrease the leaving rate of the channel for CL from CS, resulting in an increase in Po.We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cellular and Molecular Physiology, Division of Respiratory Research, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada M5G 1X8. marunaka@sickkids.on.ca

ABSTRACT
It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na+ absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na+, K+, Li+, and Cs+, but little selectivity for Ca2+ (PCa/PNa < 0.005) or Cl- (PCl/PNa < 0.01), identifying it as an NSC channel. Stretch had no additional effects on the open probability (Po) of the IBMX-activated channel. This channel had one open ("O") and two closed (short "CS" and long "CL") states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the Po of the channel without any detectable changes in the mean open or closed times. These observations led us to the conclusion that a kinetic model "CL <--> CS <--> O" was the most suitable among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of the channel for CL from CS, resulting in an increase in Po. Cytochalasin D pretreatment abolished the response to stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.

Show MeSH
Related in: MedlinePlus