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Histidine scanning mutagenesis of basic residues of the S4 segment of the shaker k+ channel.

Starace DM, Bezanilla F - J. Gen. Physiol. (2001)

Bottom Line: After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible.This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient.Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Department of Anesthesiology, University of California Los Angeles School of Medicine, Los Angeles, California 90095, USA.

ABSTRACT
The voltage sensor of the Shaker potassium channel is comprised mostly of positively charged residues in the putative fourth transmembrane segment, S4 (Aggarwal, S.K., and R. MacKinnon. 1996. Neuron. 16:1169-1177; Seoh, S.-A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Neuron. 16:1159-1167). Movement of the voltage sensor in response to a change in the membrane potential was examined indirectly by measuring how the accessibilities of residues in and around the sensor change with voltage. Each basic residue in the S4 segment was individually replaced with a histidine. If the histidine tag is part of the voltage sensor, then the gating charge displaced by the voltage sensor will include the histidine charge. Accessibility of the histidine to the bulk solution was therefore monitored as pH-dependent changes in the gating currents evoked by membrane potential pulses. Histidine scanning mutagenesis has several advantages over other similar techniques. Since histidine accessibility is detected by labeling with solution protons, very confined local environments can be resolved and labeling introduces minimal interference of voltage sensor motion. After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible. Histidine replacement of residues R365, R368, and R371, on the other hand, showed that each of these residues traverses entirely from internal exposure at hyperpolarized potentials to external exposure at depolarized potentials. This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient. In the case of 371H, depolarization drives the histidine to a position that forms a proton pore. Kinetic models of titrateable voltage sensors that account for proton transport and conduction are presented. Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure.

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Gating currents of the R368H channel displace titrateable charge and transport protons. Using the cut-open oocyte voltage clamp, R368H channel gating currents were recorded in internal HB solution, pH 7.4, and various external HB solutions (A, pHo 5.2; B, pHo 7.4; C, pHo 9.2). The intracellular pH (pHi) was measured with a H+-sensitive electrode. All gating currents were recorded from the same membrane area. The gating currents in each pHo group were elicited by a family of test pulses (in millivolts) from a pre- and postpotential pulse of −110 mV (represented at the top of each group). The test pulse value corresponding to each current is shown on the left. The normalized charge displacement in each gating current (Rel. Q, shown to the right of each trace) was obtained by integrating the OFF-gating currents (Fig. 9 B) and normalizing to the maximum value of each pHo group. Linear leak current was subtracted from each current trace off-line as described in materials and methods (Data Analysis of I-V Curves). (Experiment D11200a)
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Figure 7: Gating currents of the R368H channel displace titrateable charge and transport protons. Using the cut-open oocyte voltage clamp, R368H channel gating currents were recorded in internal HB solution, pH 7.4, and various external HB solutions (A, pHo 5.2; B, pHo 7.4; C, pHo 9.2). The intracellular pH (pHi) was measured with a H+-sensitive electrode. All gating currents were recorded from the same membrane area. The gating currents in each pHo group were elicited by a family of test pulses (in millivolts) from a pre- and postpotential pulse of −110 mV (represented at the top of each group). The test pulse value corresponding to each current is shown on the left. The normalized charge displacement in each gating current (Rel. Q, shown to the right of each trace) was obtained by integrating the OFF-gating currents (Fig. 9 B) and normalizing to the maximum value of each pHo group. Linear leak current was subtracted from each current trace off-line as described in materials and methods (Data Analysis of I-V Curves). (Experiment D11200a)

Mentions: Fig. 7 shows three series of pulse-induced gating currents from the R368H channel, each encompassing the full range of charge displacement in a different pH gradient. The pH gradients were imposed by varying the external pH (pHo) while leaving the internal solution constant. Since control of pHi is not precise in cut-open oocyte voltage clamp configuration, pHi was measured with a proton-selective microelectrode. The gating currents of the R368H channel look very different from the gating currents shown previously. The ON-gating current is a superposition of the typical transient charge displacement that decays to zero and a steady proton current transported by the histidine. In the presence of an inward proton gradient that establishes a very depolarized proton equilibrium potential, EH+ (Fig. 7 A), an inward current developed during ON-gating, increased as the membrane was increasingly depolarized, peaked at around −30 mV, and then decreased and became zero at very depolarized potentials. The size of the inward current was reduced (Fig. 7 B) when the proton gradient was reduced, and thereby decreased the proton electrochemical driving force in the voltage region of charge displacement. Reversal of the pH gradient to establish a very hyperpolarized EH+ resulted in an outward steady current also with a bell-shaped voltage dependence (Fig. 7 C). The appearance of a steady current driven by the proton electrochemical gradient only in the voltage region of frequent voltage sensor transitions indicates that it is a current of protons transported across the membrane by the histidine at position 368.


Histidine scanning mutagenesis of basic residues of the S4 segment of the shaker k+ channel.

Starace DM, Bezanilla F - J. Gen. Physiol. (2001)

Gating currents of the R368H channel displace titrateable charge and transport protons. Using the cut-open oocyte voltage clamp, R368H channel gating currents were recorded in internal HB solution, pH 7.4, and various external HB solutions (A, pHo 5.2; B, pHo 7.4; C, pHo 9.2). The intracellular pH (pHi) was measured with a H+-sensitive electrode. All gating currents were recorded from the same membrane area. The gating currents in each pHo group were elicited by a family of test pulses (in millivolts) from a pre- and postpotential pulse of −110 mV (represented at the top of each group). The test pulse value corresponding to each current is shown on the left. The normalized charge displacement in each gating current (Rel. Q, shown to the right of each trace) was obtained by integrating the OFF-gating currents (Fig. 9 B) and normalizing to the maximum value of each pHo group. Linear leak current was subtracted from each current trace off-line as described in materials and methods (Data Analysis of I-V Curves). (Experiment D11200a)
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Figure 7: Gating currents of the R368H channel displace titrateable charge and transport protons. Using the cut-open oocyte voltage clamp, R368H channel gating currents were recorded in internal HB solution, pH 7.4, and various external HB solutions (A, pHo 5.2; B, pHo 7.4; C, pHo 9.2). The intracellular pH (pHi) was measured with a H+-sensitive electrode. All gating currents were recorded from the same membrane area. The gating currents in each pHo group were elicited by a family of test pulses (in millivolts) from a pre- and postpotential pulse of −110 mV (represented at the top of each group). The test pulse value corresponding to each current is shown on the left. The normalized charge displacement in each gating current (Rel. Q, shown to the right of each trace) was obtained by integrating the OFF-gating currents (Fig. 9 B) and normalizing to the maximum value of each pHo group. Linear leak current was subtracted from each current trace off-line as described in materials and methods (Data Analysis of I-V Curves). (Experiment D11200a)
Mentions: Fig. 7 shows three series of pulse-induced gating currents from the R368H channel, each encompassing the full range of charge displacement in a different pH gradient. The pH gradients were imposed by varying the external pH (pHo) while leaving the internal solution constant. Since control of pHi is not precise in cut-open oocyte voltage clamp configuration, pHi was measured with a proton-selective microelectrode. The gating currents of the R368H channel look very different from the gating currents shown previously. The ON-gating current is a superposition of the typical transient charge displacement that decays to zero and a steady proton current transported by the histidine. In the presence of an inward proton gradient that establishes a very depolarized proton equilibrium potential, EH+ (Fig. 7 A), an inward current developed during ON-gating, increased as the membrane was increasingly depolarized, peaked at around −30 mV, and then decreased and became zero at very depolarized potentials. The size of the inward current was reduced (Fig. 7 B) when the proton gradient was reduced, and thereby decreased the proton electrochemical driving force in the voltage region of charge displacement. Reversal of the pH gradient to establish a very hyperpolarized EH+ resulted in an outward steady current also with a bell-shaped voltage dependence (Fig. 7 C). The appearance of a steady current driven by the proton electrochemical gradient only in the voltage region of frequent voltage sensor transitions indicates that it is a current of protons transported across the membrane by the histidine at position 368.

Bottom Line: After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible.This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient.Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Department of Anesthesiology, University of California Los Angeles School of Medicine, Los Angeles, California 90095, USA.

ABSTRACT
The voltage sensor of the Shaker potassium channel is comprised mostly of positively charged residues in the putative fourth transmembrane segment, S4 (Aggarwal, S.K., and R. MacKinnon. 1996. Neuron. 16:1169-1177; Seoh, S.-A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Neuron. 16:1159-1167). Movement of the voltage sensor in response to a change in the membrane potential was examined indirectly by measuring how the accessibilities of residues in and around the sensor change with voltage. Each basic residue in the S4 segment was individually replaced with a histidine. If the histidine tag is part of the voltage sensor, then the gating charge displaced by the voltage sensor will include the histidine charge. Accessibility of the histidine to the bulk solution was therefore monitored as pH-dependent changes in the gating currents evoked by membrane potential pulses. Histidine scanning mutagenesis has several advantages over other similar techniques. Since histidine accessibility is detected by labeling with solution protons, very confined local environments can be resolved and labeling introduces minimal interference of voltage sensor motion. After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible. Histidine replacement of residues R365, R368, and R371, on the other hand, showed that each of these residues traverses entirely from internal exposure at hyperpolarized potentials to external exposure at depolarized potentials. This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient. In the case of 371H, depolarization drives the histidine to a position that forms a proton pore. Kinetic models of titrateable voltage sensors that account for proton transport and conduction are presented. Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure.

Show MeSH
Related in: MedlinePlus