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pH-dependent inhibition of voltage-gated H(+) currents in rat alveolar epithelial cells by Zn(2+) and other divalent cations.

Cherny VV, DeCoursey TE - J. Gen. Physiol. (1999)

Bottom Line: Zn(2+) effects on the proton chord conductance-voltage (g(H)-V) relationship indicated higher affinities, pK(a) 7 and pK(M) 8.CdCl(2) had similar effects as ZnCl(2) and competed with H(+), but had lower affinity.Zn(2+) applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H(+) currents and slowed channel closing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA.

ABSTRACT
Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl(2) reduced the H(+) current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H(+) current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn(2+) were inconsistent with classical voltage-dependent block in which Zn(2+) binds within the membrane voltage field. Instead, Zn(2+) binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn(2+) were strongly pH(o) dependent but were insensitive to pH(i), suggesting that protons and Zn(2+) compete for external sites on H(+) channels. The apparent potency of Zn(2+) in slowing activation was approximately 10x greater at pH(o) 7 than at pH(o) 6, and approximately 100x greater at pH(o) 6 than at pH(o) 5. The pH(o) dependence suggests that Zn(2+), not ZnOH(+), is the active species. Evidently, the Zn(2+) receptor is formed by multiple groups, protonation of any of which inhibits Zn(2+) binding. The external receptor bound H(+) and Zn(2+) with pK(a) 6.2-6.6 and pK(M) 6.5, as described by several models. Zn(2+) effects on the proton chord conductance-voltage (g(H)-V) relationship indicated higher affinities, pK(a) 7 and pK(M) 8. CdCl(2) had similar effects as ZnCl(2) and competed with H(+), but had lower affinity. Zn(2+) applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H(+) currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn(2+) receptor may be the same modulatory protonation site(s) at which pH(o) regulates H(+) channel gating.

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The shift in the voltage dependence of activation of the gH produced by ZnCl2 or CdCl2 depends strongly on pHo. Mean shift ± SD are plotted for two to seven determinations in each condition (91 total). Filled symbols connected by solid lines represent ZnCl2 and open symbols with dashed lines indicate CdCl2 measurements, and the numbers indicate pHo. The shift was estimated by plotting gH-V relationships and measuring the apparent voltage shift for comparable levels of gH. The estimate was made arbitrarily when gH was large enough to be reliably determined, but always at <50% of gH,max because any reduction in gH,max (which might have a different mechanism) would contaminate the measurement. ZnCl2 and CdCl2 concentrations have been corrected for buffer binding as described in Fig. 6. The calculated unbound fraction of CdCl2 was 0.672 at pHo 7 and 0.626 at pHo 6. Because pHi had no detectable effect on externally applied metals (Fig. 6), we combined results here for pHi 5.5 and 6.5. For all data at pHo 8.0, pHi was 6.5, measurements at pHo 7 and 6 include both pHi 5.5 and 6.5, and for all data at pHo 5.5 or 5.0, pHi was 5.5.
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Figure 7: The shift in the voltage dependence of activation of the gH produced by ZnCl2 or CdCl2 depends strongly on pHo. Mean shift ± SD are plotted for two to seven determinations in each condition (91 total). Filled symbols connected by solid lines represent ZnCl2 and open symbols with dashed lines indicate CdCl2 measurements, and the numbers indicate pHo. The shift was estimated by plotting gH-V relationships and measuring the apparent voltage shift for comparable levels of gH. The estimate was made arbitrarily when gH was large enough to be reliably determined, but always at <50% of gH,max because any reduction in gH,max (which might have a different mechanism) would contaminate the measurement. ZnCl2 and CdCl2 concentrations have been corrected for buffer binding as described in Fig. 6. The calculated unbound fraction of CdCl2 was 0.672 at pHo 7 and 0.626 at pHo 6. Because pHi had no detectable effect on externally applied metals (Fig. 6), we combined results here for pHi 5.5 and 6.5. For all data at pHo 8.0, pHi was 6.5, measurements at pHo 7 and 6 include both pHi 5.5 and 6.5, and for all data at pHo 5.5 or 5.0, pHi was 5.5.

Mentions: Besides slowing activation, metals also shift channel opening to more positive voltages. This voltage shift was estimated from graphs of the gH-V relationships in the absence or presence of metal and is plotted in Fig. 7. This parameter was somewhat arbitrary and less well defined than τact, because it required extrapolating the fitted time course of H+ current and measuring Vrev in each solution (whenever pHo was changed). Nevertheless, the pHo sensitivity of the gH-V relationship to ZnCl2 (solid symbols) qualitatively resembles that of τact. In fact, the interaction between ZnCl2 and pHo manifested in the gH-V relationship appears to be somewhat stronger than that for the τact-V relationship. The concentration of ZnCl2 required to produce a 20-mV depolarizing shift of the gH-V relationship was 0.13 μM at pHo 8.0, 0.77 μM at pHo 7.0, 54 μM at pHo 6.0, 470 μM at pHo 5.5, and 12.4 mM (by extrapolation) at pHo 5.0. The apparent potency of ZnCl2 thus decreased sixfold between pHo 8 and 7, 70-fold between pHo 7 and 6, and 230-fold between pHo 6 and 5. The larger difference between the effective potency of ZnCl2 between pH 7 and pHo 8 requires a higher pKa for the steady state conductance measurement than for the kinetic τact measurement (see discussion).


pH-dependent inhibition of voltage-gated H(+) currents in rat alveolar epithelial cells by Zn(2+) and other divalent cations.

Cherny VV, DeCoursey TE - J. Gen. Physiol. (1999)

The shift in the voltage dependence of activation of the gH produced by ZnCl2 or CdCl2 depends strongly on pHo. Mean shift ± SD are plotted for two to seven determinations in each condition (91 total). Filled symbols connected by solid lines represent ZnCl2 and open symbols with dashed lines indicate CdCl2 measurements, and the numbers indicate pHo. The shift was estimated by plotting gH-V relationships and measuring the apparent voltage shift for comparable levels of gH. The estimate was made arbitrarily when gH was large enough to be reliably determined, but always at <50% of gH,max because any reduction in gH,max (which might have a different mechanism) would contaminate the measurement. ZnCl2 and CdCl2 concentrations have been corrected for buffer binding as described in Fig. 6. The calculated unbound fraction of CdCl2 was 0.672 at pHo 7 and 0.626 at pHo 6. Because pHi had no detectable effect on externally applied metals (Fig. 6), we combined results here for pHi 5.5 and 6.5. For all data at pHo 8.0, pHi was 6.5, measurements at pHo 7 and 6 include both pHi 5.5 and 6.5, and for all data at pHo 5.5 or 5.0, pHi was 5.5.
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Figure 7: The shift in the voltage dependence of activation of the gH produced by ZnCl2 or CdCl2 depends strongly on pHo. Mean shift ± SD are plotted for two to seven determinations in each condition (91 total). Filled symbols connected by solid lines represent ZnCl2 and open symbols with dashed lines indicate CdCl2 measurements, and the numbers indicate pHo. The shift was estimated by plotting gH-V relationships and measuring the apparent voltage shift for comparable levels of gH. The estimate was made arbitrarily when gH was large enough to be reliably determined, but always at <50% of gH,max because any reduction in gH,max (which might have a different mechanism) would contaminate the measurement. ZnCl2 and CdCl2 concentrations have been corrected for buffer binding as described in Fig. 6. The calculated unbound fraction of CdCl2 was 0.672 at pHo 7 and 0.626 at pHo 6. Because pHi had no detectable effect on externally applied metals (Fig. 6), we combined results here for pHi 5.5 and 6.5. For all data at pHo 8.0, pHi was 6.5, measurements at pHo 7 and 6 include both pHi 5.5 and 6.5, and for all data at pHo 5.5 or 5.0, pHi was 5.5.
Mentions: Besides slowing activation, metals also shift channel opening to more positive voltages. This voltage shift was estimated from graphs of the gH-V relationships in the absence or presence of metal and is plotted in Fig. 7. This parameter was somewhat arbitrary and less well defined than τact, because it required extrapolating the fitted time course of H+ current and measuring Vrev in each solution (whenever pHo was changed). Nevertheless, the pHo sensitivity of the gH-V relationship to ZnCl2 (solid symbols) qualitatively resembles that of τact. In fact, the interaction between ZnCl2 and pHo manifested in the gH-V relationship appears to be somewhat stronger than that for the τact-V relationship. The concentration of ZnCl2 required to produce a 20-mV depolarizing shift of the gH-V relationship was 0.13 μM at pHo 8.0, 0.77 μM at pHo 7.0, 54 μM at pHo 6.0, 470 μM at pHo 5.5, and 12.4 mM (by extrapolation) at pHo 5.0. The apparent potency of ZnCl2 thus decreased sixfold between pHo 8 and 7, 70-fold between pHo 7 and 6, and 230-fold between pHo 6 and 5. The larger difference between the effective potency of ZnCl2 between pH 7 and pHo 8 requires a higher pKa for the steady state conductance measurement than for the kinetic τact measurement (see discussion).

Bottom Line: Zn(2+) effects on the proton chord conductance-voltage (g(H)-V) relationship indicated higher affinities, pK(a) 7 and pK(M) 8.CdCl(2) had similar effects as ZnCl(2) and competed with H(+), but had lower affinity.Zn(2+) applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H(+) currents and slowed channel closing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA.

ABSTRACT
Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl(2) reduced the H(+) current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H(+) current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn(2+) were inconsistent with classical voltage-dependent block in which Zn(2+) binds within the membrane voltage field. Instead, Zn(2+) binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn(2+) were strongly pH(o) dependent but were insensitive to pH(i), suggesting that protons and Zn(2+) compete for external sites on H(+) channels. The apparent potency of Zn(2+) in slowing activation was approximately 10x greater at pH(o) 7 than at pH(o) 6, and approximately 100x greater at pH(o) 6 than at pH(o) 5. The pH(o) dependence suggests that Zn(2+), not ZnOH(+), is the active species. Evidently, the Zn(2+) receptor is formed by multiple groups, protonation of any of which inhibits Zn(2+) binding. The external receptor bound H(+) and Zn(2+) with pK(a) 6.2-6.6 and pK(M) 6.5, as described by several models. Zn(2+) effects on the proton chord conductance-voltage (g(H)-V) relationship indicated higher affinities, pK(a) 7 and pK(M) 8. CdCl(2) had similar effects as ZnCl(2) and competed with H(+), but had lower affinity. Zn(2+) applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H(+) currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn(2+) receptor may be the same modulatory protonation site(s) at which pH(o) regulates H(+) channel gating.

Show MeSH
Related in: MedlinePlus