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A novel delta current method for transport stoichiometry estimation.

Shao XM, Kao L, Kurtz I - BMC Biophys (2014)

Bottom Line: The results showed that the ΔErev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio.This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane.Computational simulations demonstrated that the ΔErev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 USA.

ABSTRACT

Background: The ion transport stoichiometry (q) of electrogenic transporters is an important determinant of their function. q can be determined by the reversal potential (Erev) if the transporter under study is the only electrogenic transport mechanism or a specific inhibitor is available. An alternative approach is to calculate delta reversal potential (ΔErev) by altering the concentrations of the transported substrates. This approach is based on the hypothesis that the contributions of other channels and transporters on the membrane to Erev are additive. However, Erev is a complicated function of the sum of different conductances rather than being additive.

Results: We propose a new delta current (ΔI) method based on a simplified model for electrogenic secondary active transport by Heinz (Electrical Potentials in Biological Membrane Transport, 1981). ΔI is the difference between two currents obtained from altering the external concentration of a transported substrate thereby eliminating other currents without the need for a specific inhibitor. q is determined by the ratio of ΔI at two different membrane voltages (V1 and V2) where q = 2RT/(F(V2 -V1))ln(ΔI2/ΔI1) + 1. We tested this ΔI methodology in HEK-293 cells expressing the elctrogenic SLC4 sodium bicarbonate cotransporters NBCe2-C and NBCe1-A, the results were consistent with those obtained with the Erev inhibitor method. Furthermore, using computational simulations, we compared the estimates of q with the ΔErev and ΔI methods. The results showed that the ΔErev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio.

Conclusions: We developed a ΔI method for estimating transport stoichiometry of electrogenic transporters based on the Heinz model. This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane. When there are other electrogenic transport pathways, ΔI method eliminates their contribution in estimating q. Computational simulations demonstrated that the ΔErev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio. This new ΔI method can be readily extended to the analysis of other electrogenic transporters in other tissues.

No MeSH data available.


Related in: MedlinePlus

HCO3−-induced current in NBCe2-C expressing HEK-293 cells. a) The cell was whole-cell voltage-clamped at -60 mV. A series of 400 ms voltage-clamp pulses range from -95 to +45 mV with increment of 10 mV were applied and whole-cell current responses were recorded. In the pre-HCO3− conditions, there is no HCO3− in the patch pipette (Table 1, patch solution a) nor in the bath solution (Table 1, Bath solution A). Increasing HCO3− concentration to 25 mM in the bath solution (Bath solution B in Table 1) induced a voltage-dependent current (central panel). The current recovered when the cell was washed with solution containing 0 HCO3− (right panel). b) Current-voltage (I-V) relation of steady-state current in the absence and presence of HCO3− (n = 8). Im (pA): membrane current in pA. Steady-state current was obtained by averaging 80 ms of the current trace toward the end of each 400 ms voltage pulse. c) I-V curve of HCO3− induced current is the difference between the I-V curves in the absence of HCO3− and in the presence of HCO3−. d) Application of 25 mM HCO3− in the bath did not induce any current in EGFP negative cells (n = 4).
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Fig2: HCO3−-induced current in NBCe2-C expressing HEK-293 cells. a) The cell was whole-cell voltage-clamped at -60 mV. A series of 400 ms voltage-clamp pulses range from -95 to +45 mV with increment of 10 mV were applied and whole-cell current responses were recorded. In the pre-HCO3− conditions, there is no HCO3− in the patch pipette (Table 1, patch solution a) nor in the bath solution (Table 1, Bath solution A). Increasing HCO3− concentration to 25 mM in the bath solution (Bath solution B in Table 1) induced a voltage-dependent current (central panel). The current recovered when the cell was washed with solution containing 0 HCO3− (right panel). b) Current-voltage (I-V) relation of steady-state current in the absence and presence of HCO3− (n = 8). Im (pA): membrane current in pA. Steady-state current was obtained by averaging 80 ms of the current trace toward the end of each 400 ms voltage pulse. c) I-V curve of HCO3− induced current is the difference between the I-V curves in the absence of HCO3− and in the presence of HCO3−. d) Application of 25 mM HCO3− in the bath did not induce any current in EGFP negative cells (n = 4).

Mentions: The light microscopic image of cultured HEK-293 cells and corresponding fluorescent image of the same field is shown in Figure 1a and b respectively. Bright fluorescent cells were EGFP positive and thus were NBCe2-C expressing cells as well. We voltage-clamped EGFP positive cells at a holding voltage -60 mV and applied a series of 400 ms pulses from -95 to +45 with increment of 10 mV. The current responses to the series of pulses in pre-HCO3− (0 HCO3−) conditions were background current due to endogenous channels in HEK-293 cells (Figure 2a left panel). We established an I-V curve of steady state current. Figure 2b shows the mean I-V curves from 8 cells. The steady state current at +45 mV was 51.8 ± 18.0 pA (mean ± SE, n = 8). Bath application of a solution containing 25 mM HCO3− (Table 1, bath solution B) induced a voltage-dependent current (Figure 2a central panel). The mean I-V curve in the presence of HCO3− is shown in Figure 2b. The steady state current at voltage +45 mV was 133.5 ± 25.5 pA (p = 0.01, paired t-test vs pre-HCO3−). The HCO3−-induced current was obtained by subtracting the current traces in the absence of HCO3− from the current traces in its presence. Figure 2c shows the mean I-V curve of HCO3− induced current. The mean HCO3−-induced current at voltage +45 mV was 81.7 ± 23.3 pA (n = 8). The current was greatly reduced after washing with the control bath solution (Figure 2a right panel). As a separate control, we tested whether the application of HCO3− containing solution induced any current in EGFP negative cells. As shown in Figure 2d, there is no significant HCO3−-induced current detected in these cells (n = 4). These results indicate that functional NBCe2-C is expressed in EGFP labeled HEK-293 cells and that NBCe2-C transports HCO3− electrogenically.Figure 1


A novel delta current method for transport stoichiometry estimation.

Shao XM, Kao L, Kurtz I - BMC Biophys (2014)

HCO3−-induced current in NBCe2-C expressing HEK-293 cells. a) The cell was whole-cell voltage-clamped at -60 mV. A series of 400 ms voltage-clamp pulses range from -95 to +45 mV with increment of 10 mV were applied and whole-cell current responses were recorded. In the pre-HCO3− conditions, there is no HCO3− in the patch pipette (Table 1, patch solution a) nor in the bath solution (Table 1, Bath solution A). Increasing HCO3− concentration to 25 mM in the bath solution (Bath solution B in Table 1) induced a voltage-dependent current (central panel). The current recovered when the cell was washed with solution containing 0 HCO3− (right panel). b) Current-voltage (I-V) relation of steady-state current in the absence and presence of HCO3− (n = 8). Im (pA): membrane current in pA. Steady-state current was obtained by averaging 80 ms of the current trace toward the end of each 400 ms voltage pulse. c) I-V curve of HCO3− induced current is the difference between the I-V curves in the absence of HCO3− and in the presence of HCO3−. d) Application of 25 mM HCO3− in the bath did not induce any current in EGFP negative cells (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4274721&req=5

Fig2: HCO3−-induced current in NBCe2-C expressing HEK-293 cells. a) The cell was whole-cell voltage-clamped at -60 mV. A series of 400 ms voltage-clamp pulses range from -95 to +45 mV with increment of 10 mV were applied and whole-cell current responses were recorded. In the pre-HCO3− conditions, there is no HCO3− in the patch pipette (Table 1, patch solution a) nor in the bath solution (Table 1, Bath solution A). Increasing HCO3− concentration to 25 mM in the bath solution (Bath solution B in Table 1) induced a voltage-dependent current (central panel). The current recovered when the cell was washed with solution containing 0 HCO3− (right panel). b) Current-voltage (I-V) relation of steady-state current in the absence and presence of HCO3− (n = 8). Im (pA): membrane current in pA. Steady-state current was obtained by averaging 80 ms of the current trace toward the end of each 400 ms voltage pulse. c) I-V curve of HCO3− induced current is the difference between the I-V curves in the absence of HCO3− and in the presence of HCO3−. d) Application of 25 mM HCO3− in the bath did not induce any current in EGFP negative cells (n = 4).
Mentions: The light microscopic image of cultured HEK-293 cells and corresponding fluorescent image of the same field is shown in Figure 1a and b respectively. Bright fluorescent cells were EGFP positive and thus were NBCe2-C expressing cells as well. We voltage-clamped EGFP positive cells at a holding voltage -60 mV and applied a series of 400 ms pulses from -95 to +45 with increment of 10 mV. The current responses to the series of pulses in pre-HCO3− (0 HCO3−) conditions were background current due to endogenous channels in HEK-293 cells (Figure 2a left panel). We established an I-V curve of steady state current. Figure 2b shows the mean I-V curves from 8 cells. The steady state current at +45 mV was 51.8 ± 18.0 pA (mean ± SE, n = 8). Bath application of a solution containing 25 mM HCO3− (Table 1, bath solution B) induced a voltage-dependent current (Figure 2a central panel). The mean I-V curve in the presence of HCO3− is shown in Figure 2b. The steady state current at voltage +45 mV was 133.5 ± 25.5 pA (p = 0.01, paired t-test vs pre-HCO3−). The HCO3−-induced current was obtained by subtracting the current traces in the absence of HCO3− from the current traces in its presence. Figure 2c shows the mean I-V curve of HCO3− induced current. The mean HCO3−-induced current at voltage +45 mV was 81.7 ± 23.3 pA (n = 8). The current was greatly reduced after washing with the control bath solution (Figure 2a right panel). As a separate control, we tested whether the application of HCO3− containing solution induced any current in EGFP negative cells. As shown in Figure 2d, there is no significant HCO3−-induced current detected in these cells (n = 4). These results indicate that functional NBCe2-C is expressed in EGFP labeled HEK-293 cells and that NBCe2-C transports HCO3− electrogenically.Figure 1

Bottom Line: The results showed that the ΔErev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio.This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane.Computational simulations demonstrated that the ΔErev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 USA.

ABSTRACT

Background: The ion transport stoichiometry (q) of electrogenic transporters is an important determinant of their function. q can be determined by the reversal potential (Erev) if the transporter under study is the only electrogenic transport mechanism or a specific inhibitor is available. An alternative approach is to calculate delta reversal potential (ΔErev) by altering the concentrations of the transported substrates. This approach is based on the hypothesis that the contributions of other channels and transporters on the membrane to Erev are additive. However, Erev is a complicated function of the sum of different conductances rather than being additive.

Results: We propose a new delta current (ΔI) method based on a simplified model for electrogenic secondary active transport by Heinz (Electrical Potentials in Biological Membrane Transport, 1981). ΔI is the difference between two currents obtained from altering the external concentration of a transported substrate thereby eliminating other currents without the need for a specific inhibitor. q is determined by the ratio of ΔI at two different membrane voltages (V1 and V2) where q = 2RT/(F(V2 -V1))ln(ΔI2/ΔI1) + 1. We tested this ΔI methodology in HEK-293 cells expressing the elctrogenic SLC4 sodium bicarbonate cotransporters NBCe2-C and NBCe1-A, the results were consistent with those obtained with the Erev inhibitor method. Furthermore, using computational simulations, we compared the estimates of q with the ΔErev and ΔI methods. The results showed that the ΔErev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio.

Conclusions: We developed a ΔI method for estimating transport stoichiometry of electrogenic transporters based on the Heinz model. This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane. When there are other electrogenic transport pathways, ΔI method eliminates their contribution in estimating q. Computational simulations demonstrated that the ΔErev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio. This new ΔI method can be readily extended to the analysis of other electrogenic transporters in other tissues.

No MeSH data available.


Related in: MedlinePlus