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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

Estimation of transport stoichiometry for NBCe2-C using conventional reversal potential method. a) In the conditions of equal concentrations (25 mM) of HCO3− intra- and extracellularly, the ratio of intracellular concentration of Na+ ([Na+]i) and extracellular concentration of Na+ ([Na+]o) = 40/80 mM (Patch solution d/bath solution C in Table 1), cells were voltage-clamped at -60 mV. Current responses to a series of 400 mV voltage pulses from -95 to +45 mV with increment of 10 mV were recorded in the absence (pre-DIDS Control, Ctrl) and presence of DIDS (0.5 mM). DIDS sensitive current (right panel, Ctrl-DIDS) was obtained by digital subtraction of currents in the presence of DIDS (center panel) from control current (n = 3). B) I-V relations of steady-state current in Ctrl, DIDS and Ctrl-DIDS conditions. c) I-V curves obtained with the same protocol as (b) except [Na+]i/[Na+]o = 25/135 mM (Patch solution c/bath solution B in Table 1) (n = 5). d) The two VI=0 values are close to the calculated values assuming q = 2 (dash lines) while significantly distinct from the calculated values assuming q = 3 (dash lines).
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Fig3: Estimation of transport stoichiometry for NBCe2-C using conventional reversal potential method. a) In the conditions of equal concentrations (25 mM) of HCO3− intra- and extracellularly, the ratio of intracellular concentration of Na+ ([Na+]i) and extracellular concentration of Na+ ([Na+]o) = 40/80 mM (Patch solution d/bath solution C in Table 1), cells were voltage-clamped at -60 mV. Current responses to a series of 400 mV voltage pulses from -95 to +45 mV with increment of 10 mV were recorded in the absence (pre-DIDS Control, Ctrl) and presence of DIDS (0.5 mM). DIDS sensitive current (right panel, Ctrl-DIDS) was obtained by digital subtraction of currents in the presence of DIDS (center panel) from control current (n = 3). B) I-V relations of steady-state current in Ctrl, DIDS and Ctrl-DIDS conditions. c) I-V curves obtained with the same protocol as (b) except [Na+]i/[Na+]o = 25/135 mM (Patch solution c/bath solution B in Table 1) (n = 5). d) The two VI=0 values are close to the calculated values assuming q = 2 (dash lines) while significantly distinct from the calculated values assuming q = 3 (dash lines).

Mentions: In this study, HEK-293 cells expressing NBCe2-C were whole-cell patch-clamped at -60 mV. VI=0 was measured in two independent experiments where [HCO3−]i and [HCO3−]o were equal (25 mM), therefore ENBC depended only on [Na+]i/[Na+]o. For every cell recorded, we waited at least 10 min from establishment of whole-cell patch-clamp to ensure that [Na+]i and [HCO3−]i were equal to the concentrations of Na+ and HCO3− respectively in the patch pipette solution by diffusion before beginning I-V measurement. Current responses to a series of voltage pulses were recorded to establish I-V relationship in the absence and presence of DIDS (0.5 mM, Figure 3a). In the first experiment, using [Na+]i/[Na+]o = 40/80 mM (Patch solution d/bath solution C in Table 1), I-V curve of steady-state NBCe2-C transport current (DIDS sensitive current) was obtained by subtraction of currents in the presence of DIDS from control current (pre-DIDS). VI=0 = -22.3 ± 2.4 mV (n = 3) was obtained (Figure 3a,b and d). To show the mean and variability among cells, this VI=0 value was averaged from the VI=0 of individual sample cells. Note that this mean VI=0 value is very close to the VI=0 points where the average DIDS-sensitive I-V curve crosses the x-axis in (Figure 3b). In the second experiment using [Na+]i/[Na+]o = 25/135 mM (Patch solution c/bath solution B in Table 1), we got VI=0 = -43.9 ± 3.5 mV (n = 5, Figure 3c and d). The two VI=0 values are close to the calculated ENBC values of -17.8 and -43.3 mV (Eq. 1), respectively, assuming q = 2 (dash lines) while significantly distinct from the calculated values assuming q = 3 (dash lines, Figure 3d). The results indicate that the transport stoichiometry ratio of NBCe2-C is 2 HCO3−: 1 Na+ or (1 CO32−: 1 Na+) in HEK-293 cells.Figure 3


A novel delta current method for transport stoichiometry estimation.

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

Estimation of transport stoichiometry for NBCe2-C using conventional reversal potential method. a) In the conditions of equal concentrations (25 mM) of HCO3− intra- and extracellularly, the ratio of intracellular concentration of Na+ ([Na+]i) and extracellular concentration of Na+ ([Na+]o) = 40/80 mM (Patch solution d/bath solution C in Table 1), cells were voltage-clamped at -60 mV. Current responses to a series of 400 mV voltage pulses from -95 to +45 mV with increment of 10 mV were recorded in the absence (pre-DIDS Control, Ctrl) and presence of DIDS (0.5 mM). DIDS sensitive current (right panel, Ctrl-DIDS) was obtained by digital subtraction of currents in the presence of DIDS (center panel) from control current (n = 3). B) I-V relations of steady-state current in Ctrl, DIDS and Ctrl-DIDS conditions. c) I-V curves obtained with the same protocol as (b) except [Na+]i/[Na+]o = 25/135 mM (Patch solution c/bath solution B in Table 1) (n = 5). d) The two VI=0 values are close to the calculated values assuming q = 2 (dash lines) while significantly distinct from the calculated values assuming q = 3 (dash lines).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Estimation of transport stoichiometry for NBCe2-C using conventional reversal potential method. a) In the conditions of equal concentrations (25 mM) of HCO3− intra- and extracellularly, the ratio of intracellular concentration of Na+ ([Na+]i) and extracellular concentration of Na+ ([Na+]o) = 40/80 mM (Patch solution d/bath solution C in Table 1), cells were voltage-clamped at -60 mV. Current responses to a series of 400 mV voltage pulses from -95 to +45 mV with increment of 10 mV were recorded in the absence (pre-DIDS Control, Ctrl) and presence of DIDS (0.5 mM). DIDS sensitive current (right panel, Ctrl-DIDS) was obtained by digital subtraction of currents in the presence of DIDS (center panel) from control current (n = 3). B) I-V relations of steady-state current in Ctrl, DIDS and Ctrl-DIDS conditions. c) I-V curves obtained with the same protocol as (b) except [Na+]i/[Na+]o = 25/135 mM (Patch solution c/bath solution B in Table 1) (n = 5). d) The two VI=0 values are close to the calculated values assuming q = 2 (dash lines) while significantly distinct from the calculated values assuming q = 3 (dash lines).
Mentions: In this study, HEK-293 cells expressing NBCe2-C were whole-cell patch-clamped at -60 mV. VI=0 was measured in two independent experiments where [HCO3−]i and [HCO3−]o were equal (25 mM), therefore ENBC depended only on [Na+]i/[Na+]o. For every cell recorded, we waited at least 10 min from establishment of whole-cell patch-clamp to ensure that [Na+]i and [HCO3−]i were equal to the concentrations of Na+ and HCO3− respectively in the patch pipette solution by diffusion before beginning I-V measurement. Current responses to a series of voltage pulses were recorded to establish I-V relationship in the absence and presence of DIDS (0.5 mM, Figure 3a). In the first experiment, using [Na+]i/[Na+]o = 40/80 mM (Patch solution d/bath solution C in Table 1), I-V curve of steady-state NBCe2-C transport current (DIDS sensitive current) was obtained by subtraction of currents in the presence of DIDS from control current (pre-DIDS). VI=0 = -22.3 ± 2.4 mV (n = 3) was obtained (Figure 3a,b and d). To show the mean and variability among cells, this VI=0 value was averaged from the VI=0 of individual sample cells. Note that this mean VI=0 value is very close to the VI=0 points where the average DIDS-sensitive I-V curve crosses the x-axis in (Figure 3b). In the second experiment using [Na+]i/[Na+]o = 25/135 mM (Patch solution c/bath solution B in Table 1), we got VI=0 = -43.9 ± 3.5 mV (n = 5, Figure 3c and d). The two VI=0 values are close to the calculated ENBC values of -17.8 and -43.3 mV (Eq. 1), respectively, assuming q = 2 (dash lines) while significantly distinct from the calculated values assuming q = 3 (dash lines, Figure 3d). The results indicate that the transport stoichiometry ratio of NBCe2-C is 2 HCO3−: 1 Na+ or (1 CO32−: 1 Na+) in HEK-293 cells.Figure 3

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