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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.


Estimation of transport stoichiometry for NBCe2-C using the delta current method. a) NBCe2-C expressing cells were voltage clamped at -50 mV. A series of 400 ms voltage-clamp pulses range from -108 to +48 mV with increment of 12 mV (containing a pulse to 0 mV during this protocol) was applied and whole-cell current responses were recorded. Patch pipette solution contained 10 mM Na+ and 25 mM HCO3− (Solution b in Table 1). Bath solution also contained 10 mM Na+ and 25 mM HCO3− (Bath solution D in Table 1) (left panel). Enhancing Na+ concentration from 10 to 25 mM in the bath solution (Bath solution E in Table 1) increased voltage-dependent current (central panel). Net current (ΔI) through NBCe2-C induced by changing [Na+]o is obtained by subtracting the current traces at [Na+]o = 10 mM from the current traces at [Na+]o = 25 mM (right panel). b) Current-voltage (I-V) relations of steady-state current (mean of 80 ms current trace toward the end of each voltage pulse) in bath solutions containing 10 mM and 25 mM Na+. c) I-V relation of ΔI. d) Estimation of transport stoichiometry ratio q with Eq. 7 (n = 5).
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Fig4: Estimation of transport stoichiometry for NBCe2-C using the delta current method. a) NBCe2-C expressing cells were voltage clamped at -50 mV. A series of 400 ms voltage-clamp pulses range from -108 to +48 mV with increment of 12 mV (containing a pulse to 0 mV during this protocol) was applied and whole-cell current responses were recorded. Patch pipette solution contained 10 mM Na+ and 25 mM HCO3− (Solution b in Table 1). Bath solution also contained 10 mM Na+ and 25 mM HCO3− (Bath solution D in Table 1) (left panel). Enhancing Na+ concentration from 10 to 25 mM in the bath solution (Bath solution E in Table 1) increased voltage-dependent current (central panel). Net current (ΔI) through NBCe2-C induced by changing [Na+]o is obtained by subtracting the current traces at [Na+]o = 10 mM from the current traces at [Na+]o = 25 mM (right panel). b) Current-voltage (I-V) relations of steady-state current (mean of 80 ms current trace toward the end of each voltage pulse) in bath solutions containing 10 mM and 25 mM Na+. c) I-V relation of ΔI. d) Estimation of transport stoichiometry ratio q with Eq. 7 (n = 5).

Mentions: Under the conditions that [Na+]i = [Na+]o = 10 mM and [HCO3−]i = [HCO3−]o = 25 mM (patch solution b and bath solution D in Table 1), NBCe2-C expressing HEK-293 cells were voltage-clamped at -50 mV and a series of voltage (including a pulse to 0 mV) was applied (Figure 4a, left panel). Increasing the Na+ concentration from 10 to 25 mM in the bath solution (bath solution E in Table 1) increased the voltage-dependent current (Figure 4a, central panel). Net current (ΔI) through NBCe2-C induced by changing [Na+]o was obtained by subtracting the currents in bath solution containing 10 mM Na+ from currents in 25 mM [Na+]o (Figure 4a, right panel). With this operation, according to Eq. 4, currents mediated by other channels and electrogenic transporters were eliminated if the two assumptions associated with Eq. 4 were satisfied. Figure 4b shows current-voltage (I-V) relation of steady-state current in bath solution containing 10 mM or 25 mM [Na+]o and Figure 4c shows ΔI of NBCe2-C vs. voltages. Taking ΔIv1 at V = 0 and ΔIv2 at V = 12 mV, q is calculated using Eq. 7. We obtained q = 2.0 ± 0.14 (n = 5, Figure 4d). The results suggest that the transport stoichiometry ratio of NBCe2-C is 2 HCO3−: 1 Na+ (or 1 CO32−: 1 Na+) in HEK-293 cells. This result is consistent with the q value obtained with the conventional reversal potential method using the inhibitor DIDS (Figure 3).Figure 4


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 the delta current method. a) NBCe2-C expressing cells were voltage clamped at -50 mV. A series of 400 ms voltage-clamp pulses range from -108 to +48 mV with increment of 12 mV (containing a pulse to 0 mV during this protocol) was applied and whole-cell current responses were recorded. Patch pipette solution contained 10 mM Na+ and 25 mM HCO3− (Solution b in Table 1). Bath solution also contained 10 mM Na+ and 25 mM HCO3− (Bath solution D in Table 1) (left panel). Enhancing Na+ concentration from 10 to 25 mM in the bath solution (Bath solution E in Table 1) increased voltage-dependent current (central panel). Net current (ΔI) through NBCe2-C induced by changing [Na+]o is obtained by subtracting the current traces at [Na+]o = 10 mM from the current traces at [Na+]o = 25 mM (right panel). b) Current-voltage (I-V) relations of steady-state current (mean of 80 ms current trace toward the end of each voltage pulse) in bath solutions containing 10 mM and 25 mM Na+. c) I-V relation of ΔI. d) Estimation of transport stoichiometry ratio q with Eq. 7 (n = 5).
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Related In: Results  -  Collection

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Fig4: Estimation of transport stoichiometry for NBCe2-C using the delta current method. a) NBCe2-C expressing cells were voltage clamped at -50 mV. A series of 400 ms voltage-clamp pulses range from -108 to +48 mV with increment of 12 mV (containing a pulse to 0 mV during this protocol) was applied and whole-cell current responses were recorded. Patch pipette solution contained 10 mM Na+ and 25 mM HCO3− (Solution b in Table 1). Bath solution also contained 10 mM Na+ and 25 mM HCO3− (Bath solution D in Table 1) (left panel). Enhancing Na+ concentration from 10 to 25 mM in the bath solution (Bath solution E in Table 1) increased voltage-dependent current (central panel). Net current (ΔI) through NBCe2-C induced by changing [Na+]o is obtained by subtracting the current traces at [Na+]o = 10 mM from the current traces at [Na+]o = 25 mM (right panel). b) Current-voltage (I-V) relations of steady-state current (mean of 80 ms current trace toward the end of each voltage pulse) in bath solutions containing 10 mM and 25 mM Na+. c) I-V relation of ΔI. d) Estimation of transport stoichiometry ratio q with Eq. 7 (n = 5).
Mentions: Under the conditions that [Na+]i = [Na+]o = 10 mM and [HCO3−]i = [HCO3−]o = 25 mM (patch solution b and bath solution D in Table 1), NBCe2-C expressing HEK-293 cells were voltage-clamped at -50 mV and a series of voltage (including a pulse to 0 mV) was applied (Figure 4a, left panel). Increasing the Na+ concentration from 10 to 25 mM in the bath solution (bath solution E in Table 1) increased the voltage-dependent current (Figure 4a, central panel). Net current (ΔI) through NBCe2-C induced by changing [Na+]o was obtained by subtracting the currents in bath solution containing 10 mM Na+ from currents in 25 mM [Na+]o (Figure 4a, right panel). With this operation, according to Eq. 4, currents mediated by other channels and electrogenic transporters were eliminated if the two assumptions associated with Eq. 4 were satisfied. Figure 4b shows current-voltage (I-V) relation of steady-state current in bath solution containing 10 mM or 25 mM [Na+]o and Figure 4c shows ΔI of NBCe2-C vs. voltages. Taking ΔIv1 at V = 0 and ΔIv2 at V = 12 mV, q is calculated using Eq. 7. We obtained q = 2.0 ± 0.14 (n = 5, Figure 4d). The results suggest that the transport stoichiometry ratio of NBCe2-C is 2 HCO3−: 1 Na+ (or 1 CO32−: 1 Na+) in HEK-293 cells. This result is consistent with the q value obtained with the conventional reversal potential method using the inhibitor DIDS (Figure 3).Figure 4

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.