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New insights on the voltage dependence of the KCa3.1 channel block by internal TBA.

Banderali U, Klein H, Garneau L, Simoes M, Parent L, Sauvé R - J. Gen. Physiol. (2004)

Bottom Line: The model also indicates that raising the internal K+ concentration should decrease the value of delta measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect delta for concentrations >50 mM.All these predictions are born out by our current experimental results.Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter.

View Article: PubMed Central - PubMed

Affiliation: Département de Physiologie, Membrane Protein Study Group, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

ABSTRACT
We present in this work a structural model of the open IKCa (KCa3.1) channel derived by homology modeling from the MthK channel structure, and used this model to compute the transmembrane potential profile along the channel pore. This analysis showed that the selectivity filter and the region extending from the channel inner cavity to the internal medium should respectively account for 81% and 16% of the transmembrane potential difference. We found however that the voltage dependence of the IKCa block by the quaternary ammonium ion TBA applied internally is compatible with an apparent electrical distance delta of 0.49 +/- 0.02 (n = 6) for negative potentials. To reconcile this observation with the electrostatic potential profile predicted for the channel pore, we modeled the IKCa block by TBA assuming that the voltage dependence of the block is governed by both the difference in potential between the channel cavity and the internal medium, and the potential profile along the selectivity filter region through an effect on the filter ion occupancy states. The resulting model predicts that delta should be voltage dependent, being larger at negative than positive potentials. The model also indicates that raising the internal K+ concentration should decrease the value of delta measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect delta for concentrations >50 mM. All these predictions are born out by our current experimental results. Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter. Globally, these results support a model whereby the voltage dependence of the TBA block in IKCa is mainly governed by the voltage dependence of the ion occupancy states of the selectivity filter.

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Voltage dependence of the TBA-induced IKCa channel block. (A) Dose–response curves of IKCa block by TBA measured in EBIO (100 μM) conditions at membrane potentials ranging from −120 to −30 mV. The concentration for half inhibition IC50 was computed by fitting the experimental data to Eq. 1. Increasing hyperpolarizing voltages rightward shifted the dose–response curves with IC50 values of 666 ± 17 μM (n = 6) and 113 ± 4 μM (n = 6) at −120 and −30 mV, respectively. (B) Plot of IC50 as a function of voltage measured in the presence (filled squares) and in the absence (open squares) of EBIO. Data points within the voltage range −120 to −30 mV were fitted to a single exponential function resulting in an electrical distance of 0.49 ± 0.02 (n = 6). The continuous lines were computed from the model presented in Fig. 6 A using Eqs. 13 and 14 with IC50 = <KB4>/<KB3>. The best fit was obtained with KB41(0) = 8 s−1, KB42(0) = 28 s−1, KB43(0) = 1.7 s−1, KB31(0) = 0.022 s−1μM−1, KB32(0) = 0.07 s−1μM−1, KB33(0) = 0.1 s−1μM−1 with EBIO and KB41(0) = 2.9 s−1, KB42(0) = 10.4 s−1, KB43(0) = 0.63 s−1, KB31(0) = 0.006 s−1μM−1, KB32(0) = 0.02 s−1μM−1, KB33(0) = 0.026 s−1μM−1 without EBIO. (C) Plot of apparent TBA exit rate ζPoK4 as a function of voltage measured either in the presence (filled squares) or in the absence (open squares) of EBIO. EBIO elicited an average 2.7-fold increase of the TBA exit rates. For voltages ranging from −150 to −30 mV, the apparent exit rate ζPoK4 decreased an e-fold factor per 90 mV for a voltage dependence with an electrical distance of 0.26 ± 0.02. The continuous lines represent the prediction of the model of Fig. 6 A computed according to Eq. 14 with KB41(0), KB42(0), and KB43(0) as in B. (D) Plot of the apparent TBA entry rate PoK3 as a function of voltage measured either in the presence (filled squares) or in the absence (open squares) of EBIO. Filled (EBIO) and open (no EBIO) diamonds show TBA entry rates K3 after correcting for the channel open probabilities Po. The resulting K3 values are shown to be comparable under both experimental conditions with a voltage dependence corresponding to an e-fold increase per 150 mV. The continuous lines represent the prediction of the model of Fig. 6 A, computed according to Eq. 13 with KB31(0), KB32(0), and KB33(0) as in B.
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fig6: Voltage dependence of the TBA-induced IKCa channel block. (A) Dose–response curves of IKCa block by TBA measured in EBIO (100 μM) conditions at membrane potentials ranging from −120 to −30 mV. The concentration for half inhibition IC50 was computed by fitting the experimental data to Eq. 1. Increasing hyperpolarizing voltages rightward shifted the dose–response curves with IC50 values of 666 ± 17 μM (n = 6) and 113 ± 4 μM (n = 6) at −120 and −30 mV, respectively. (B) Plot of IC50 as a function of voltage measured in the presence (filled squares) and in the absence (open squares) of EBIO. Data points within the voltage range −120 to −30 mV were fitted to a single exponential function resulting in an electrical distance of 0.49 ± 0.02 (n = 6). The continuous lines were computed from the model presented in Fig. 6 A using Eqs. 13 and 14 with IC50 = <KB4>/<KB3>. The best fit was obtained with KB41(0) = 8 s−1, KB42(0) = 28 s−1, KB43(0) = 1.7 s−1, KB31(0) = 0.022 s−1μM−1, KB32(0) = 0.07 s−1μM−1, KB33(0) = 0.1 s−1μM−1 with EBIO and KB41(0) = 2.9 s−1, KB42(0) = 10.4 s−1, KB43(0) = 0.63 s−1, KB31(0) = 0.006 s−1μM−1, KB32(0) = 0.02 s−1μM−1, KB33(0) = 0.026 s−1μM−1 without EBIO. (C) Plot of apparent TBA exit rate ζPoK4 as a function of voltage measured either in the presence (filled squares) or in the absence (open squares) of EBIO. EBIO elicited an average 2.7-fold increase of the TBA exit rates. For voltages ranging from −150 to −30 mV, the apparent exit rate ζPoK4 decreased an e-fold factor per 90 mV for a voltage dependence with an electrical distance of 0.26 ± 0.02. The continuous lines represent the prediction of the model of Fig. 6 A computed according to Eq. 14 with KB41(0), KB42(0), and KB43(0) as in B. (D) Plot of the apparent TBA entry rate PoK3 as a function of voltage measured either in the presence (filled squares) or in the absence (open squares) of EBIO. Filled (EBIO) and open (no EBIO) diamonds show TBA entry rates K3 after correcting for the channel open probabilities Po. The resulting K3 values are shown to be comparable under both experimental conditions with a voltage dependence corresponding to an e-fold increase per 150 mV. The continuous lines represent the prediction of the model of Fig. 6 A, computed according to Eq. 13 with KB31(0), KB32(0), and KB33(0) as in B.

Mentions: Fig. 5 A summarizes the effect of voltage on the TBA dose–response curve for voltages ranging from −30 to −120 mV, measured in EBIO conditions. As seen, negative membrane voltages resulted in a rightward shift of the TBA dose–response curve. The variation in IC50 as a function of voltage is presented in Fig. 5 B. Clearly, the voltage dependence of the TBA block shows a complex behavior with a stronger voltage dependence at negative than at positive potentials. In the former case, the data could be approximated to a single exponential function of the form IC50 = IC50(0) exp(−δVq/KT), where δ is the fraction of the transmembrane potential through which internal TBA moves to reach its site (Woodhull, 1973), V the applied voltage, q, K, and T the electrical charge, the Boltzmann constant, and the temperature, respectively. Estimations of the electrical distance δ within the voltage range −120 to −30 mV led to a value of 0.49 ± 0.02 (n = 6).


New insights on the voltage dependence of the KCa3.1 channel block by internal TBA.

Banderali U, Klein H, Garneau L, Simoes M, Parent L, Sauvé R - J. Gen. Physiol. (2004)

Voltage dependence of the TBA-induced IKCa channel block. (A) Dose–response curves of IKCa block by TBA measured in EBIO (100 μM) conditions at membrane potentials ranging from −120 to −30 mV. The concentration for half inhibition IC50 was computed by fitting the experimental data to Eq. 1. Increasing hyperpolarizing voltages rightward shifted the dose–response curves with IC50 values of 666 ± 17 μM (n = 6) and 113 ± 4 μM (n = 6) at −120 and −30 mV, respectively. (B) Plot of IC50 as a function of voltage measured in the presence (filled squares) and in the absence (open squares) of EBIO. Data points within the voltage range −120 to −30 mV were fitted to a single exponential function resulting in an electrical distance of 0.49 ± 0.02 (n = 6). The continuous lines were computed from the model presented in Fig. 6 A using Eqs. 13 and 14 with IC50 = <KB4>/<KB3>. The best fit was obtained with KB41(0) = 8 s−1, KB42(0) = 28 s−1, KB43(0) = 1.7 s−1, KB31(0) = 0.022 s−1μM−1, KB32(0) = 0.07 s−1μM−1, KB33(0) = 0.1 s−1μM−1 with EBIO and KB41(0) = 2.9 s−1, KB42(0) = 10.4 s−1, KB43(0) = 0.63 s−1, KB31(0) = 0.006 s−1μM−1, KB32(0) = 0.02 s−1μM−1, KB33(0) = 0.026 s−1μM−1 without EBIO. (C) Plot of apparent TBA exit rate ζPoK4 as a function of voltage measured either in the presence (filled squares) or in the absence (open squares) of EBIO. EBIO elicited an average 2.7-fold increase of the TBA exit rates. For voltages ranging from −150 to −30 mV, the apparent exit rate ζPoK4 decreased an e-fold factor per 90 mV for a voltage dependence with an electrical distance of 0.26 ± 0.02. The continuous lines represent the prediction of the model of Fig. 6 A computed according to Eq. 14 with KB41(0), KB42(0), and KB43(0) as in B. (D) Plot of the apparent TBA entry rate PoK3 as a function of voltage measured either in the presence (filled squares) or in the absence (open squares) of EBIO. Filled (EBIO) and open (no EBIO) diamonds show TBA entry rates K3 after correcting for the channel open probabilities Po. The resulting K3 values are shown to be comparable under both experimental conditions with a voltage dependence corresponding to an e-fold increase per 150 mV. The continuous lines represent the prediction of the model of Fig. 6 A, computed according to Eq. 13 with KB31(0), KB32(0), and KB33(0) as in B.
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fig6: Voltage dependence of the TBA-induced IKCa channel block. (A) Dose–response curves of IKCa block by TBA measured in EBIO (100 μM) conditions at membrane potentials ranging from −120 to −30 mV. The concentration for half inhibition IC50 was computed by fitting the experimental data to Eq. 1. Increasing hyperpolarizing voltages rightward shifted the dose–response curves with IC50 values of 666 ± 17 μM (n = 6) and 113 ± 4 μM (n = 6) at −120 and −30 mV, respectively. (B) Plot of IC50 as a function of voltage measured in the presence (filled squares) and in the absence (open squares) of EBIO. Data points within the voltage range −120 to −30 mV were fitted to a single exponential function resulting in an electrical distance of 0.49 ± 0.02 (n = 6). The continuous lines were computed from the model presented in Fig. 6 A using Eqs. 13 and 14 with IC50 = <KB4>/<KB3>. The best fit was obtained with KB41(0) = 8 s−1, KB42(0) = 28 s−1, KB43(0) = 1.7 s−1, KB31(0) = 0.022 s−1μM−1, KB32(0) = 0.07 s−1μM−1, KB33(0) = 0.1 s−1μM−1 with EBIO and KB41(0) = 2.9 s−1, KB42(0) = 10.4 s−1, KB43(0) = 0.63 s−1, KB31(0) = 0.006 s−1μM−1, KB32(0) = 0.02 s−1μM−1, KB33(0) = 0.026 s−1μM−1 without EBIO. (C) Plot of apparent TBA exit rate ζPoK4 as a function of voltage measured either in the presence (filled squares) or in the absence (open squares) of EBIO. EBIO elicited an average 2.7-fold increase of the TBA exit rates. For voltages ranging from −150 to −30 mV, the apparent exit rate ζPoK4 decreased an e-fold factor per 90 mV for a voltage dependence with an electrical distance of 0.26 ± 0.02. The continuous lines represent the prediction of the model of Fig. 6 A computed according to Eq. 14 with KB41(0), KB42(0), and KB43(0) as in B. (D) Plot of the apparent TBA entry rate PoK3 as a function of voltage measured either in the presence (filled squares) or in the absence (open squares) of EBIO. Filled (EBIO) and open (no EBIO) diamonds show TBA entry rates K3 after correcting for the channel open probabilities Po. The resulting K3 values are shown to be comparable under both experimental conditions with a voltage dependence corresponding to an e-fold increase per 150 mV. The continuous lines represent the prediction of the model of Fig. 6 A, computed according to Eq. 13 with KB31(0), KB32(0), and KB33(0) as in B.
Mentions: Fig. 5 A summarizes the effect of voltage on the TBA dose–response curve for voltages ranging from −30 to −120 mV, measured in EBIO conditions. As seen, negative membrane voltages resulted in a rightward shift of the TBA dose–response curve. The variation in IC50 as a function of voltage is presented in Fig. 5 B. Clearly, the voltage dependence of the TBA block shows a complex behavior with a stronger voltage dependence at negative than at positive potentials. In the former case, the data could be approximated to a single exponential function of the form IC50 = IC50(0) exp(−δVq/KT), where δ is the fraction of the transmembrane potential through which internal TBA moves to reach its site (Woodhull, 1973), V the applied voltage, q, K, and T the electrical charge, the Boltzmann constant, and the temperature, respectively. Estimations of the electrical distance δ within the voltage range −120 to −30 mV led to a value of 0.49 ± 0.02 (n = 6).

Bottom Line: The model also indicates that raising the internal K+ concentration should decrease the value of delta measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect delta for concentrations >50 mM.All these predictions are born out by our current experimental results.Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter.

View Article: PubMed Central - PubMed

Affiliation: Département de Physiologie, Membrane Protein Study Group, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

ABSTRACT
We present in this work a structural model of the open IKCa (KCa3.1) channel derived by homology modeling from the MthK channel structure, and used this model to compute the transmembrane potential profile along the channel pore. This analysis showed that the selectivity filter and the region extending from the channel inner cavity to the internal medium should respectively account for 81% and 16% of the transmembrane potential difference. We found however that the voltage dependence of the IKCa block by the quaternary ammonium ion TBA applied internally is compatible with an apparent electrical distance delta of 0.49 +/- 0.02 (n = 6) for negative potentials. To reconcile this observation with the electrostatic potential profile predicted for the channel pore, we modeled the IKCa block by TBA assuming that the voltage dependence of the block is governed by both the difference in potential between the channel cavity and the internal medium, and the potential profile along the selectivity filter region through an effect on the filter ion occupancy states. The resulting model predicts that delta should be voltage dependent, being larger at negative than positive potentials. The model also indicates that raising the internal K+ concentration should decrease the value of delta measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect delta for concentrations >50 mM. All these predictions are born out by our current experimental results. Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter. Globally, these results support a model whereby the voltage dependence of the TBA block in IKCa is mainly governed by the voltage dependence of the ion occupancy states of the selectivity filter.

Show MeSH
Related in: MedlinePlus