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Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel.

Rothberg BS, Shin KS, Phale PS, Yellen G - J. Gen. Physiol. (2002)

Bottom Line: The rate of this recovery also was reduced when channels were held at depolarized voltages.Thus, Cd(2+) escape is also gated at the intracellular side of the channel.Together, these results are consistent with a voltage-controlled structure at the intracellular side of the spHCN channel that can gate the flow of cations through the pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.

ABSTRACT
Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Our previous work using the specific HCN channel blocker ZD7288 provided evidence for an intracellular activation gate for these channels because it appears that ZD7288, applied from the intracellular side, can enter and leave HCN channels only at voltages where the activation gate is opened (Shin, K.S., B.S. Rothberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91-101). However, the ZD7288 molecule is larger than the Na(+) or K(+) ions that flow through the open channel. In the present study, we sought to resolve whether the voltage gate at the intracellular entrance to the pore for ZD7288 also can be a gate for permeant ions in HCN channels. Single residues in the putative pore-lining S6 region of an HCN channel (cloned from sea urchin; spHCN) were substituted with cysteines, and the mutants were probed with Cd(2+) applied to the intracellular side of the channel. One mutant, T464C, displayed rapid irreversible block when Cd(2+) was applied to opened channels, with an apparent blocking rate of approximately 3 x 10(5) M(-1)s(-1). The blocking rate was decreased for channels held at more depolarized voltages that close the channels, which is consistent with the Cd(2+) access to this residue being gated from the intracellular side of the channel. 464C channels could be recovered from Cd(2+) inhibition in the presence of a dithiol applied to the intracellular side. The rate of this recovery also was reduced when channels were held at depolarized voltages. Finally, Cd(2+) could be trapped inside channels that were composed of WT/464C tandem-linked subunits, which could otherwise recover spontaneously from Cd(2+) inhibition. Thus, Cd(2+) escape is also gated at the intracellular side of the channel. Together, these results are consistent with a voltage-controlled structure at the intracellular side of the spHCN channel that can gate the flow of cations through the pore.

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Cd2+ inhibition is reversible in channels containing only two cysteines at position 464. (A) Reversible Cd2+ inhibition in channels composed of TC tandem-linked subunit dimers (results). The TC/TC channels were ∼80% inhibited in 6 μM Cd2+, and the current recovered to near control levels in ∼2 min. The channels were ∼86% inhibited during a subsequent application of 20 μM Cd2+. (B) Cd2+ inhibition in patches containing a mixture of channels composed of TT and CC dimers (results). In this experiment, the initial application of 20 μM Cd2+ inhibited ∼90% of the current (CC/CC + TT/CC channels). Upon removal of Cd2+, ∼25% of the current recovered in ∼2 min (TT/CC channels). 65% of the current was blocked irreversibly (CC/CC channels). The recovered 25% could again be reversibly blocked by a subsequent application of 20 μM Cd2+. The remaining 10% of the current was unaffected by Cd2+ (mostly TT/TT channels). For both A and B, channels were held at +10 mV, and current was monitored using 400-ms test pulses to −110 mV (closed circles). Linear leak current was subtracted, and currents were normalized to the mean pre-Cd2+ control level.
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Figure 4: Cd2+ inhibition is reversible in channels containing only two cysteines at position 464. (A) Reversible Cd2+ inhibition in channels composed of TC tandem-linked subunit dimers (results). The TC/TC channels were ∼80% inhibited in 6 μM Cd2+, and the current recovered to near control levels in ∼2 min. The channels were ∼86% inhibited during a subsequent application of 20 μM Cd2+. (B) Cd2+ inhibition in patches containing a mixture of channels composed of TT and CC dimers (results). In this experiment, the initial application of 20 μM Cd2+ inhibited ∼90% of the current (CC/CC + TT/CC channels). Upon removal of Cd2+, ∼25% of the current recovered in ∼2 min (TT/CC channels). 65% of the current was blocked irreversibly (CC/CC channels). The recovered 25% could again be reversibly blocked by a subsequent application of 20 μM Cd2+. The remaining 10% of the current was unaffected by Cd2+ (mostly TT/TT channels). For both A and B, channels were held at +10 mV, and current was monitored using 400-ms test pulses to −110 mV (closed circles). Linear leak current was subtracted, and currents were normalized to the mean pre-Cd2+ control level.

Mentions: We introduced the 464C mutation into one protomer in a tandem dimer construct; the other protomer contained the wild-type residue T464 (dimer TC). If channels are always formed by two tandem dimers (TC/TC), then these channels will contain two cysteines at 464 instead of four. In patches excised from cells transfected with the TC construct, 20 μM Cd2+ inhibited >80% of the current, but this effect was almost completely reversible upon washout (Fig. 4 A). This suggests that a channel containing two cysteines at 464 can be at least partially inhibited by Cd, but that two cysteines are not sufficient to bind Cd2+ irreversibly.


Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel.

Rothberg BS, Shin KS, Phale PS, Yellen G - J. Gen. Physiol. (2002)

Cd2+ inhibition is reversible in channels containing only two cysteines at position 464. (A) Reversible Cd2+ inhibition in channels composed of TC tandem-linked subunit dimers (results). The TC/TC channels were ∼80% inhibited in 6 μM Cd2+, and the current recovered to near control levels in ∼2 min. The channels were ∼86% inhibited during a subsequent application of 20 μM Cd2+. (B) Cd2+ inhibition in patches containing a mixture of channels composed of TT and CC dimers (results). In this experiment, the initial application of 20 μM Cd2+ inhibited ∼90% of the current (CC/CC + TT/CC channels). Upon removal of Cd2+, ∼25% of the current recovered in ∼2 min (TT/CC channels). 65% of the current was blocked irreversibly (CC/CC channels). The recovered 25% could again be reversibly blocked by a subsequent application of 20 μM Cd2+. The remaining 10% of the current was unaffected by Cd2+ (mostly TT/TT channels). For both A and B, channels were held at +10 mV, and current was monitored using 400-ms test pulses to −110 mV (closed circles). Linear leak current was subtracted, and currents were normalized to the mean pre-Cd2+ control level.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233860&req=5

Figure 4: Cd2+ inhibition is reversible in channels containing only two cysteines at position 464. (A) Reversible Cd2+ inhibition in channels composed of TC tandem-linked subunit dimers (results). The TC/TC channels were ∼80% inhibited in 6 μM Cd2+, and the current recovered to near control levels in ∼2 min. The channels were ∼86% inhibited during a subsequent application of 20 μM Cd2+. (B) Cd2+ inhibition in patches containing a mixture of channels composed of TT and CC dimers (results). In this experiment, the initial application of 20 μM Cd2+ inhibited ∼90% of the current (CC/CC + TT/CC channels). Upon removal of Cd2+, ∼25% of the current recovered in ∼2 min (TT/CC channels). 65% of the current was blocked irreversibly (CC/CC channels). The recovered 25% could again be reversibly blocked by a subsequent application of 20 μM Cd2+. The remaining 10% of the current was unaffected by Cd2+ (mostly TT/TT channels). For both A and B, channels were held at +10 mV, and current was monitored using 400-ms test pulses to −110 mV (closed circles). Linear leak current was subtracted, and currents were normalized to the mean pre-Cd2+ control level.
Mentions: We introduced the 464C mutation into one protomer in a tandem dimer construct; the other protomer contained the wild-type residue T464 (dimer TC). If channels are always formed by two tandem dimers (TC/TC), then these channels will contain two cysteines at 464 instead of four. In patches excised from cells transfected with the TC construct, 20 μM Cd2+ inhibited >80% of the current, but this effect was almost completely reversible upon washout (Fig. 4 A). This suggests that a channel containing two cysteines at 464 can be at least partially inhibited by Cd, but that two cysteines are not sufficient to bind Cd2+ irreversibly.

Bottom Line: The rate of this recovery also was reduced when channels were held at depolarized voltages.Thus, Cd(2+) escape is also gated at the intracellular side of the channel.Together, these results are consistent with a voltage-controlled structure at the intracellular side of the spHCN channel that can gate the flow of cations through the pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.

ABSTRACT
Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Our previous work using the specific HCN channel blocker ZD7288 provided evidence for an intracellular activation gate for these channels because it appears that ZD7288, applied from the intracellular side, can enter and leave HCN channels only at voltages where the activation gate is opened (Shin, K.S., B.S. Rothberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91-101). However, the ZD7288 molecule is larger than the Na(+) or K(+) ions that flow through the open channel. In the present study, we sought to resolve whether the voltage gate at the intracellular entrance to the pore for ZD7288 also can be a gate for permeant ions in HCN channels. Single residues in the putative pore-lining S6 region of an HCN channel (cloned from sea urchin; spHCN) were substituted with cysteines, and the mutants were probed with Cd(2+) applied to the intracellular side of the channel. One mutant, T464C, displayed rapid irreversible block when Cd(2+) was applied to opened channels, with an apparent blocking rate of approximately 3 x 10(5) M(-1)s(-1). The blocking rate was decreased for channels held at more depolarized voltages that close the channels, which is consistent with the Cd(2+) access to this residue being gated from the intracellular side of the channel. 464C channels could be recovered from Cd(2+) inhibition in the presence of a dithiol applied to the intracellular side. The rate of this recovery also was reduced when channels were held at depolarized voltages. Finally, Cd(2+) could be trapped inside channels that were composed of WT/464C tandem-linked subunits, which could otherwise recover spontaneously from Cd(2+) inhibition. Thus, Cd(2+) escape is also gated at the intracellular side of the channel. Together, these results are consistent with a voltage-controlled structure at the intracellular side of the spHCN channel that can gate the flow of cations through the pore.

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Related in: MedlinePlus