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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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The BLM K+ channel is a true inward rectifier. Representative current records at various command potentials (−Vpip) from an inside-out basolateral membrane patch in symmetrical [K] containing at least three K+ channels. The patch pipette and bath each contain 95 mM K+ (solution d) with 0.2 mM ATP added to the bath. The dashed line represents the all channels closed (leak) current at each potential. Each open channel level is denoted by a dotted line. (B) Effect of [Mg2+]i on the I-V relation of the BLM K+ channel. The inward rectification evident in 1 mM [Mg2+]i (○) is relieved when [Mg2+]i is lowered to 200 nM (▪). (inset) Slope conductance–voltage (g-V) relation for the BLM K+ channel in 1 mM [Mg2+]i (○) and 200 nM [Mg2+]i (▪). Symbols represent mean ± SEM. The K+ channel prefers Tl+ over K+. The I-V relation from inside-out patches with Tl-acetate (solution g) in the pipette and K-acetate (solution h) in the bath is also shown (▴). The limiting inward slope conductance is 29.0 ± 1.0 pS (n = 4) for Tl+ compared with 24.5 ± 0.6 pS (n = 8) for K+. (C) Channel activity (nPo) increases with hyperpolarization. Data from four inside-out membrane patches in symmetrical [K] are plotted. Channel activity at command potentials of −120, −100, −80, −60, and −40 mV was normalized to that at −100 mV for comparison. Solid line is a single exponential fit with a voltage constant of ∼83 mV. Symbols represent mean (•) ± SEM (bars).
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Figure 3: The BLM K+ channel is a true inward rectifier. Representative current records at various command potentials (−Vpip) from an inside-out basolateral membrane patch in symmetrical [K] containing at least three K+ channels. The patch pipette and bath each contain 95 mM K+ (solution d) with 0.2 mM ATP added to the bath. The dashed line represents the all channels closed (leak) current at each potential. Each open channel level is denoted by a dotted line. (B) Effect of [Mg2+]i on the I-V relation of the BLM K+ channel. The inward rectification evident in 1 mM [Mg2+]i (○) is relieved when [Mg2+]i is lowered to 200 nM (▪). (inset) Slope conductance–voltage (g-V) relation for the BLM K+ channel in 1 mM [Mg2+]i (○) and 200 nM [Mg2+]i (▪). Symbols represent mean ± SEM. The K+ channel prefers Tl+ over K+. The I-V relation from inside-out patches with Tl-acetate (solution g) in the pipette and K-acetate (solution h) in the bath is also shown (▴). The limiting inward slope conductance is 29.0 ± 1.0 pS (n = 4) for Tl+ compared with 24.5 ± 0.6 pS (n = 8) for K+. (C) Channel activity (nPo) increases with hyperpolarization. Data from four inside-out membrane patches in symmetrical [K] are plotted. Channel activity at command potentials of −120, −100, −80, −60, and −40 mV was normalized to that at −100 mV for comparison. Solid line is a single exponential fit with a voltage constant of ∼83 mV. Symbols represent mean (•) ± SEM (bars).

Mentions: Measurements of single-channel current with [K+] = 95 mM on both sides of the membrane patch (plus 0.2 mM ATP on the cytosolic side) demonstrate that the BLM K+ channel is a true inward rectifier (Fig. 3, A and B). The I-V relation in symmetrical [K+] inwardly rectifies and reverses very close to EK = 0 mV. The channel has an inward slope conductance of γslope, in = 24.5 ± 0.6 pS (n = 8, measured between −60 and −100 mV), and an inward chord conductance of γchord, in = 20.5 ± 0.4 pS (n = 8, measured between Erev = 0 and −100 mV). The outward chord conductance measured at +80 mV is γchord, out = 3.7 ± 0.4 pS (n = 2). The outward slope conductance between +20 and +80 mV is clearly smaller.


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)

The BLM K+ channel is a true inward rectifier. Representative current records at various command potentials (−Vpip) from an inside-out basolateral membrane patch in symmetrical [K] containing at least three K+ channels. The patch pipette and bath each contain 95 mM K+ (solution d) with 0.2 mM ATP added to the bath. The dashed line represents the all channels closed (leak) current at each potential. Each open channel level is denoted by a dotted line. (B) Effect of [Mg2+]i on the I-V relation of the BLM K+ channel. The inward rectification evident in 1 mM [Mg2+]i (○) is relieved when [Mg2+]i is lowered to 200 nM (▪). (inset) Slope conductance–voltage (g-V) relation for the BLM K+ channel in 1 mM [Mg2+]i (○) and 200 nM [Mg2+]i (▪). Symbols represent mean ± SEM. The K+ channel prefers Tl+ over K+. The I-V relation from inside-out patches with Tl-acetate (solution g) in the pipette and K-acetate (solution h) in the bath is also shown (▴). The limiting inward slope conductance is 29.0 ± 1.0 pS (n = 4) for Tl+ compared with 24.5 ± 0.6 pS (n = 8) for K+. (C) Channel activity (nPo) increases with hyperpolarization. Data from four inside-out membrane patches in symmetrical [K] are plotted. Channel activity at command potentials of −120, −100, −80, −60, and −40 mV was normalized to that at −100 mV for comparison. Solid line is a single exponential fit with a voltage constant of ∼83 mV. Symbols represent mean (•) ± SEM (bars).
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Figure 3: The BLM K+ channel is a true inward rectifier. Representative current records at various command potentials (−Vpip) from an inside-out basolateral membrane patch in symmetrical [K] containing at least three K+ channels. The patch pipette and bath each contain 95 mM K+ (solution d) with 0.2 mM ATP added to the bath. The dashed line represents the all channels closed (leak) current at each potential. Each open channel level is denoted by a dotted line. (B) Effect of [Mg2+]i on the I-V relation of the BLM K+ channel. The inward rectification evident in 1 mM [Mg2+]i (○) is relieved when [Mg2+]i is lowered to 200 nM (▪). (inset) Slope conductance–voltage (g-V) relation for the BLM K+ channel in 1 mM [Mg2+]i (○) and 200 nM [Mg2+]i (▪). Symbols represent mean ± SEM. The K+ channel prefers Tl+ over K+. The I-V relation from inside-out patches with Tl-acetate (solution g) in the pipette and K-acetate (solution h) in the bath is also shown (▴). The limiting inward slope conductance is 29.0 ± 1.0 pS (n = 4) for Tl+ compared with 24.5 ± 0.6 pS (n = 8) for K+. (C) Channel activity (nPo) increases with hyperpolarization. Data from four inside-out membrane patches in symmetrical [K] are plotted. Channel activity at command potentials of −120, −100, −80, −60, and −40 mV was normalized to that at −100 mV for comparison. Solid line is a single exponential fit with a voltage constant of ∼83 mV. Symbols represent mean (•) ± SEM (bars).
Mentions: Measurements of single-channel current with [K+] = 95 mM on both sides of the membrane patch (plus 0.2 mM ATP on the cytosolic side) demonstrate that the BLM K+ channel is a true inward rectifier (Fig. 3, A and B). The I-V relation in symmetrical [K+] inwardly rectifies and reverses very close to EK = 0 mV. The channel has an inward slope conductance of γslope, in = 24.5 ± 0.6 pS (n = 8, measured between −60 and −100 mV), and an inward chord conductance of γchord, in = 20.5 ± 0.4 pS (n = 8, measured between Erev = 0 and −100 mV). The outward chord conductance measured at +80 mV is γchord, out = 3.7 ± 0.4 pS (n = 2). The outward slope conductance between +20 and +80 mV is clearly smaller.

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