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Localization of the activation gate of a voltage-gated Ca2+ channel.

Xie C, Zhen XG, Yang J - J. Gen. Physiol. (2005)

Bottom Line: We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state.In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states.Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K+ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca2+ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the alpha1 subunit of P/Q type Ca2+ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.

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Trapping of MTSET in the inner pore in the closed state. (A) Two voltage protocols for measuring MTSET modification. Currents were evoked by a depolarizing pulse to +30 mV every 6 s from a holding potential of −80 mV. The duration of the depolarizing pulse was either 500 ms or 10 ms. (B) Time course of MTSET inhibition of currents evoked by the 500-ms test pulse (filled circles) or the 10-ms test pulse (open circles) in the IIS6-A4C mutant channel. (C) The MTSET inhibition phase in B plotted against the cumulative channel open time, superimposed with a single-exponential fitting curve with the indicated time constant.
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fig7: Trapping of MTSET in the inner pore in the closed state. (A) Two voltage protocols for measuring MTSET modification. Currents were evoked by a depolarizing pulse to +30 mV every 6 s from a holding potential of −80 mV. The duration of the depolarizing pulse was either 500 ms or 10 ms. (B) Time course of MTSET inhibition of currents evoked by the 500-ms test pulse (filled circles) or the 10-ms test pulse (open circles) in the IIS6-A4C mutant channel. (C) The MTSET inhibition phase in B plotted against the cumulative channel open time, superimposed with a single-exponential fitting curve with the indicated time constant.

Mentions: In many channels, blocking molecules are often trapped in the pore when the channel is closed (e.g., Armstrong, 1966, 1971, 1974; Holmgren et al., 1997; Shin et al., 2001; for review see Hille 2001), a phenomenon indicative of an intracellular gate. Does trapping occur to MTSET in VGCCs? To address this question, we compared MTSET modification of IIS6-A4C using two different voltage protocols, in which the magnitude of the test pulse (+30 mV) and the pulse interval (6 s) were maintained the same but the duration of the test pulse was set at 500 ms in one case and 10 ms in another (Fig. 7 A). IIS6-A4C was chosen because it exhibited little modification in the closed state (Fig. 5 A). This mutant channel was modified with both voltage protocols, but the modification was much faster with the 500-ms test pulse than with the 10-ms test pulse for the same number of test pulses (Fig. 7 B). This is expected if modification occurs predominantly in the open state. However, when the time course of modification was plotted using the cumulative channel open time, it became apparent that the modification was significantly faster (approximately fourfold) with the 10-ms test pulse (Fig. 7 C). One potential factor contributing to this apparent faster modification is the time (2–3 ms) taken for the open channels to completely close, which would increase the actual cumulative channel open time for the 10-ms test pulse by 20–30% (2–3 ms adds little to the 500-ms test pulse). However, this addition would increase the modification rate by only 20–30%. Furthermore, since 2–3 ms was also needed for all the channels to open, the real open time for the 10-ms test pulse should remain more or less 10 ms. A more likely explanation is trapping: MTSET enters the inner pore when the channel is open; it then gets trapped in the inner pore when an intracellular gate is closed and reacts with the cysteine while the channel remains in the closed state. Since more trapping events occur with the 10-ms test pulse for the same cumulative channel open time, the apparent modification rate becomes faster. Trapping in another way supports the existence of an intracellular gate.


Localization of the activation gate of a voltage-gated Ca2+ channel.

Xie C, Zhen XG, Yang J - J. Gen. Physiol. (2005)

Trapping of MTSET in the inner pore in the closed state. (A) Two voltage protocols for measuring MTSET modification. Currents were evoked by a depolarizing pulse to +30 mV every 6 s from a holding potential of −80 mV. The duration of the depolarizing pulse was either 500 ms or 10 ms. (B) Time course of MTSET inhibition of currents evoked by the 500-ms test pulse (filled circles) or the 10-ms test pulse (open circles) in the IIS6-A4C mutant channel. (C) The MTSET inhibition phase in B plotted against the cumulative channel open time, superimposed with a single-exponential fitting curve with the indicated time constant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266579&req=5

fig7: Trapping of MTSET in the inner pore in the closed state. (A) Two voltage protocols for measuring MTSET modification. Currents were evoked by a depolarizing pulse to +30 mV every 6 s from a holding potential of −80 mV. The duration of the depolarizing pulse was either 500 ms or 10 ms. (B) Time course of MTSET inhibition of currents evoked by the 500-ms test pulse (filled circles) or the 10-ms test pulse (open circles) in the IIS6-A4C mutant channel. (C) The MTSET inhibition phase in B plotted against the cumulative channel open time, superimposed with a single-exponential fitting curve with the indicated time constant.
Mentions: In many channels, blocking molecules are often trapped in the pore when the channel is closed (e.g., Armstrong, 1966, 1971, 1974; Holmgren et al., 1997; Shin et al., 2001; for review see Hille 2001), a phenomenon indicative of an intracellular gate. Does trapping occur to MTSET in VGCCs? To address this question, we compared MTSET modification of IIS6-A4C using two different voltage protocols, in which the magnitude of the test pulse (+30 mV) and the pulse interval (6 s) were maintained the same but the duration of the test pulse was set at 500 ms in one case and 10 ms in another (Fig. 7 A). IIS6-A4C was chosen because it exhibited little modification in the closed state (Fig. 5 A). This mutant channel was modified with both voltage protocols, but the modification was much faster with the 500-ms test pulse than with the 10-ms test pulse for the same number of test pulses (Fig. 7 B). This is expected if modification occurs predominantly in the open state. However, when the time course of modification was plotted using the cumulative channel open time, it became apparent that the modification was significantly faster (approximately fourfold) with the 10-ms test pulse (Fig. 7 C). One potential factor contributing to this apparent faster modification is the time (2–3 ms) taken for the open channels to completely close, which would increase the actual cumulative channel open time for the 10-ms test pulse by 20–30% (2–3 ms adds little to the 500-ms test pulse). However, this addition would increase the modification rate by only 20–30%. Furthermore, since 2–3 ms was also needed for all the channels to open, the real open time for the 10-ms test pulse should remain more or less 10 ms. A more likely explanation is trapping: MTSET enters the inner pore when the channel is open; it then gets trapped in the inner pore when an intracellular gate is closed and reacts with the cysteine while the channel remains in the closed state. Since more trapping events occur with the 10-ms test pulse for the same cumulative channel open time, the apparent modification rate becomes faster. Trapping in another way supports the existence of an intracellular gate.

Bottom Line: We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state.In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states.Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K+ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca2+ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the alpha1 subunit of P/Q type Ca2+ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.

Show MeSH
Related in: MedlinePlus