ATP Dependence of the ICl, swell Channel Varies with Rate of Cell Swelling Evidence for Two Modes of Channel Activation

Bond, Tamara; Basavappa, Srisaila; Christensen, Michael; Strange, Kevin

doi:10.1085/jgp.113.3.441

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Article| March 01 1999

ATP Dependence of the I_{Cl, swell} Channel Varies with Rate of Cell Swelling : Evidence for Two Modes of Channel Activation

Tamara Bond,

Tamara Bond

From the *Department of Anesthesiology and ‡Department of Pharmacology, Anesthesiology Research Division, Vanderbilt University Medical Center, Nashville, Tennessee 37232

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Srisaila Basavappa,

Srisaila Basavappa

From the *Department of Anesthesiology and ‡Department of Pharmacology, Anesthesiology Research Division, Vanderbilt University Medical Center, Nashville, Tennessee 37232

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Michael Christensen,

Michael Christensen

From the *Department of Anesthesiology and ‡Department of Pharmacology, Anesthesiology Research Division, Vanderbilt University Medical Center, Nashville, Tennessee 37232

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Kevin Strange

From the *Department of Anesthesiology and ‡Department of Pharmacology, Anesthesiology Research Division, Vanderbilt University Medical Center, Nashville, Tennessee 37232

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Author and Article Information

Tamara Bond , Srisaila Basavappa , Michael Christensen , Kevin Strange

From the *Department of Anesthesiology and ‡Department of Pharmacology, Anesthesiology Research Division, Vanderbilt University Medical Center, Nashville, Tennessee 37232

Address correspondence to Dr. Kevin Strange, Department of Anesthesiology, Vanderbilt University Medical Center, 504 Oxford House, 1313 21st Avenue South, Nashville, TN 37232. Fax: 615-343-3916; E-mail: [email protected]

Dr. Basavappa's current address is Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520.

2

A plot of the data in Table I revealed that there was a linear relationship between the rate of cell swelling and reductions in bath osmolality of 50–200 mOsm. However, the line intersected the x axis at ∼20 mOsm, instead of 0 mOsm as expected. This suggests that there is a resistance to cell swelling with osmotic gradients <20 mOsm. The reason for this resistance to swelling with small hypotonic shocks is uncertain. It should be emphasized, however, that it does not effect the interpretation of results presented in this paper.

3

It is noteworthy that the effect of a high rate of cell swelling on ATP sensitivity was altered by intracellular solution composition. During the course of these studies, the cells developed a nonselective cation conductance that interfered with measurements of I_{Cl, swell}. This conductance was eliminated by replacing CsCl in either the bath alone, or in both the bath and patch pipette solution with NMDG-Cl. However, in the presence of intracellular NMDG-Cl, ATP-independent activation of I_{Cl, swell} did not occur when cells were swollen by exposure to a 150-mOsm reduction in bath osmolality (data not shown). The nature of this effect of intracellular NMDG-Cl is unknown.

4

It was necessary to reduce bath NMDG-Cl concentration from 70 to 40 mM to reduce the osmolality by 200 mOsm. The mean ± SEM E_rev of the current observed in cells exposed to this solution was 22.3 ± 0.8 mV (n = 7). The expected E_rev calculated from the Goldman-Hodgkin-Katz equation is 25.1 mV. In experiments where bath NMDG-Cl concentration was 70 mM, whole cell currents were quantified at +60 mV, which is 47 mV above the calculated E_rev. Therefore, whole cell currents were quantified at +72 mV in cells bathed with 40 mM NMDG-Cl.

Control experiments were performed to determine if the reduction in bath salt concentration altered current activation. Cells were exposed to a bath solution containing 40 mM NMDG-Cl and osmolality was reduced by 150 mOsm. The mean ± SEM rate of current activation in these cells was 4.8 ± 1.4 pA/pF per min (n = 3). This value was not significantly different (P > 0.1) from that observed in cells swollen in the presence of 70 mM NMDG-Cl.

Received: October 05 1998

Accepted: January 13 1999

Online ISSN: 1540-7748

Print ISSN: 0022-1295

1999

J Gen Physiol (1999) 113 (3): 441–456.

https://doi.org/10.1085/jgp.113.3.441

Swelling-induced activation of the outwardly rectifying anion current, I_{Cl, swell}, is modulated by intracellular ATP. The mechanisms by which ATP controls channel activation, however, are unknown. Whole cell patch clamp was employed to begin addressing this issue. Endogenous ATP production was inhibited by dialyzing N1E115 neuroblastoma cells for 4–5 min with solutions containing (μM): 40 oligomycin, 5 iodoacetate, and 20 rotenone. The effect of ATP on current activation was observed in the absence of intracellular Mg²⁺, in cells exposed to extracellular metabolic inhibitors for 25–35 min followed by intracellular dialysis with oligomycin, iodoacetate, and rotenone, after substitution of ATP with the nonhydrolyzable analogue AMP-PNP, and in the presence of AMP-PNP and alkaline phosphatase to dephosphorylate intracellular proteins. These results demonstrate that the ATP dependence of the channel requires ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15–55%/min in the absence of intracellular ATP, current activation was slow (0.3–0.8 pA/pF per min). ATP concentration increased the rate of current activation up to maximal values of 4–6 pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a saturable, hyperbolic function of ATP concentration. The EC₅₀ for ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4–6 pA/pF per min) in the absence of ATP when cells were swollen at rates ≥65%/min. Intracellular ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous ATP was dialyzed out of the cytoplasm of cells swollen at 15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory proteins are capable of sensing the rate of cell volume increase. We suggest that channel activation occurs via ATP-dependent and -independent mechanisms. Increasing the rate of cell swelling appears to increase the proportion of channels activating via the ATP-independent pathway. These findings have important physiological implications for understanding I_{Cl, swell} regulation, the mechanisms by which cells sense volume changes, and volume homeostasis under conditions where cell metabolism is compromised.

1999

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ATP Dependence of the I_{Cl, swell} Channel Varies with Rate of Cell Swelling : Evidence for Two Modes of Channel Activation