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Properties of an inwardly rectifying ATP-sensitive K+ channel in the basolateral membrane of renal proximal tubule.

Mauerer UR, Boulpaep EL, Segal AS - J. Gen. Physiol. (1998)

Bottom Line: The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+, Cs+, Li+, NH4+, or Cl-.The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP.We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na+,K+-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K+ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K+ channels (gammaslope, in = 24.5 +/- 0.6 pS, gammachord, out = 3.7 +/- 0.4 pS). The rectification is mediated in part by internal Mg2+. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding-unbinding model. The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+, Cs+, Li+, NH4+, or Cl-. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0. 2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.

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ATP but not ATP-γS prevents and rescues channel rundown. Hydrolyzable nucleoside triphosphates prevent and rescue BLM K+ channel rundown. A running average (current versus time, window width 768 ms) of a representative experiment is shown. The pipette is KCl (solution d), the bath is NaCl (solution c), the command potential is −60 mV. Upon excision in a nucleotide-free bath, channel activity decreases (channel rundown). After the addition of 0.2 mM ATP to the bath, channel activity slowly recovers. The addition of 0.2 mM ATP-γS (a poorly hydrolyzable ATP analogue) in the continued presence of 0.2 mM ATP has no effect. However, when ATP is removed, ATP-γS is not able to support channel activity, which rapidly declines and runs down. Readdition of ATP leads to full recovery of channel activity. Single-channel traces showing that ATP-γS has an inhibitory effect on KATP channel activity in excised inside-out BLM patches. When compared with control conditions (top), the addition of ATP-γS (middle) reduces nPo. This inhibition is reversible as long as the exposure to ATP-γS is not prolonged (bottom). (C) Removal of Mg2+ does not prevent channel rundown in an ATP-free bath. The top panel shows that rundown of the BLM KATP channel upon excision into an ATP-free bath proceeds despite removal of bath Mg2+. Representative traces from the regions marked by α, β, and γ are shown at bottom.
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Figure 9: ATP but not ATP-γS prevents and rescues channel rundown. Hydrolyzable nucleoside triphosphates prevent and rescue BLM K+ channel rundown. A running average (current versus time, window width 768 ms) of a representative experiment is shown. The pipette is KCl (solution d), the bath is NaCl (solution c), the command potential is −60 mV. Upon excision in a nucleotide-free bath, channel activity decreases (channel rundown). After the addition of 0.2 mM ATP to the bath, channel activity slowly recovers. The addition of 0.2 mM ATP-γS (a poorly hydrolyzable ATP analogue) in the continued presence of 0.2 mM ATP has no effect. However, when ATP is removed, ATP-γS is not able to support channel activity, which rapidly declines and runs down. Readdition of ATP leads to full recovery of channel activity. Single-channel traces showing that ATP-γS has an inhibitory effect on KATP channel activity in excised inside-out BLM patches. When compared with control conditions (top), the addition of ATP-γS (middle) reduces nPo. This inhibition is reversible as long as the exposure to ATP-γS is not prolonged (bottom). (C) Removal of Mg2+ does not prevent channel rundown in an ATP-free bath. The top panel shows that rundown of the BLM KATP channel upon excision into an ATP-free bath proceeds despite removal of bath Mg2+. Representative traces from the regions marked by α, β, and γ are shown at bottom.

Mentions: One characteristic of KATP channels is “rundown,” a gradual loss of activity when the membrane patch is deprived of cytosolic ATP (Findlay and Dunne, 1986). Typically, both Mg2+ and ATP are required to prevent rundown in KATP channels (Ashcroft and Ashcroft, 1990). Likewise, the BLM K+ channel runs down in the absence of either Mg2+ or ATP (or both). Lower concentrations of ATP (100–200 μM) will prevent or “rescue” channel rundown. The experiment shown in Fig. 9A summarizes the characteristics of BLM K+ channel rundown. Channel activity typically begins to decrease (rundown) upon excision of the membrane patch into a nucleotide-free bath. If this process is allowed to continue, channel activity will cease, usually irreversibly. When 0.2 mM of ATP is added back, channel activity can be restored. When ATP is removed, all channels rapidly close. In the continued presence of ATP-γS, readdition of ATP is again able to rescue rundown, and activity returns to baseline upon washout of the ATP-γS. Frequently (but not invariably), ATP-γS has an inhibitory effect on single channel activity when added in the presence of ATP, which is reversible as long as the exposure is not prolonged (n = 6), as shown in Fig. 9B. When ATP-γS is added in the absence of ATP, channel activity runs down very quickly, usually irreversibly (n = 4, data not shown).


Properties of an inwardly rectifying ATP-sensitive K+ channel in the basolateral membrane of renal proximal tubule.

Mauerer UR, Boulpaep EL, Segal AS - J. Gen. Physiol. (1998)

ATP but not ATP-γS prevents and rescues channel rundown. Hydrolyzable nucleoside triphosphates prevent and rescue BLM K+ channel rundown. A running average (current versus time, window width 768 ms) of a representative experiment is shown. The pipette is KCl (solution d), the bath is NaCl (solution c), the command potential is −60 mV. Upon excision in a nucleotide-free bath, channel activity decreases (channel rundown). After the addition of 0.2 mM ATP to the bath, channel activity slowly recovers. The addition of 0.2 mM ATP-γS (a poorly hydrolyzable ATP analogue) in the continued presence of 0.2 mM ATP has no effect. However, when ATP is removed, ATP-γS is not able to support channel activity, which rapidly declines and runs down. Readdition of ATP leads to full recovery of channel activity. Single-channel traces showing that ATP-γS has an inhibitory effect on KATP channel activity in excised inside-out BLM patches. When compared with control conditions (top), the addition of ATP-γS (middle) reduces nPo. This inhibition is reversible as long as the exposure to ATP-γS is not prolonged (bottom). (C) Removal of Mg2+ does not prevent channel rundown in an ATP-free bath. The top panel shows that rundown of the BLM KATP channel upon excision into an ATP-free bath proceeds despite removal of bath Mg2+. Representative traces from the regions marked by α, β, and γ are shown at bottom.
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Related In: Results  -  Collection

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Figure 9: ATP but not ATP-γS prevents and rescues channel rundown. Hydrolyzable nucleoside triphosphates prevent and rescue BLM K+ channel rundown. A running average (current versus time, window width 768 ms) of a representative experiment is shown. The pipette is KCl (solution d), the bath is NaCl (solution c), the command potential is −60 mV. Upon excision in a nucleotide-free bath, channel activity decreases (channel rundown). After the addition of 0.2 mM ATP to the bath, channel activity slowly recovers. The addition of 0.2 mM ATP-γS (a poorly hydrolyzable ATP analogue) in the continued presence of 0.2 mM ATP has no effect. However, when ATP is removed, ATP-γS is not able to support channel activity, which rapidly declines and runs down. Readdition of ATP leads to full recovery of channel activity. Single-channel traces showing that ATP-γS has an inhibitory effect on KATP channel activity in excised inside-out BLM patches. When compared with control conditions (top), the addition of ATP-γS (middle) reduces nPo. This inhibition is reversible as long as the exposure to ATP-γS is not prolonged (bottom). (C) Removal of Mg2+ does not prevent channel rundown in an ATP-free bath. The top panel shows that rundown of the BLM KATP channel upon excision into an ATP-free bath proceeds despite removal of bath Mg2+. Representative traces from the regions marked by α, β, and γ are shown at bottom.
Mentions: One characteristic of KATP channels is “rundown,” a gradual loss of activity when the membrane patch is deprived of cytosolic ATP (Findlay and Dunne, 1986). Typically, both Mg2+ and ATP are required to prevent rundown in KATP channels (Ashcroft and Ashcroft, 1990). Likewise, the BLM K+ channel runs down in the absence of either Mg2+ or ATP (or both). Lower concentrations of ATP (100–200 μM) will prevent or “rescue” channel rundown. The experiment shown in Fig. 9A summarizes the characteristics of BLM K+ channel rundown. Channel activity typically begins to decrease (rundown) upon excision of the membrane patch into a nucleotide-free bath. If this process is allowed to continue, channel activity will cease, usually irreversibly. When 0.2 mM of ATP is added back, channel activity can be restored. When ATP is removed, all channels rapidly close. In the continued presence of ATP-γS, readdition of ATP is again able to rescue rundown, and activity returns to baseline upon washout of the ATP-γS. Frequently (but not invariably), ATP-γS has an inhibitory effect on single channel activity when added in the presence of ATP, which is reversible as long as the exposure is not prolonged (n = 6), as shown in Fig. 9B. When ATP-γS is added in the absence of ATP, channel activity runs down very quickly, usually irreversibly (n = 4, data not shown).

Bottom Line: The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+, Cs+, Li+, NH4+, or Cl-.The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP.We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na+,K+-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K+ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K+ channels (gammaslope, in = 24.5 +/- 0.6 pS, gammachord, out = 3.7 +/- 0.4 pS). The rectification is mediated in part by internal Mg2+. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding-unbinding model. The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+, Cs+, Li+, NH4+, or Cl-. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0. 2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.

Show MeSH
Related in: MedlinePlus