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Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel.

Obejero-Paz CA, Gray IP, Jones SW - J. Gen. Physiol. (2004)

Bottom Line: Closed channels were also blocked by Y(3+) at a concentration-dependent rate, only eightfold slower than open-channel block.When extracellular Ca(2+) was replaced with Ba(2+), the rate of open block by Y(3+) was unaffected, but closed block was threefold faster than in Ca(2+), suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate.Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular "pore gate" has also been proposed. We have used the voltage dependence of block by extracellular Y(3+) as a tool to locate the activation gate of the alpha1G (Ca(V)3.1) T-type calcium channel. Y(3+) block exhibited no clear voltage dependence from -40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y(3+) at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca(2+) was replaced with Ba(2+), the rate of open block by Y(3+) was unaffected, but closed block was threefold faster than in Ca(2+), suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.

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Reentry of Y3+ into open channels. (A) Effect of 1 μM Y3+ on tail currents. A 1-ms step to +200 mV was used to both activate channels and relieve Y3+ block. Currents are shown following repolarization to +80 mV, −40 mV, and −160 mV. Currents at +200 mV are off scale. 1.5 kHz Gaussian filter. (B) Acceleration of tail currents by Y3+. Tail currents were fitted by single exponentials in control conditions, and in four concentrations of Y3+. Values are not shown at +20 mV, near the reversal potential. n = 4–6. (C) Voltage- and concentration-dependent reblocking. For each cell, at each voltage, the blocking rate was calculated from tail current time constants in Y3+ and in control conditions (Eq. 1).
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fig4: Reentry of Y3+ into open channels. (A) Effect of 1 μM Y3+ on tail currents. A 1-ms step to +200 mV was used to both activate channels and relieve Y3+ block. Currents are shown following repolarization to +80 mV, −40 mV, and −160 mV. Currents at +200 mV are off scale. 1.5 kHz Gaussian filter. (B) Acceleration of tail currents by Y3+. Tail currents were fitted by single exponentials in control conditions, and in four concentrations of Y3+. Values are not shown at +20 mV, near the reversal potential. n = 4–6. (C) Voltage- and concentration-dependent reblocking. For each cell, at each voltage, the blocking rate was calculated from tail current time constants in Y3+ and in control conditions (Eq. 1).

Mentions: To examine reblocking by Y3+, channels were first unblocked by a 1-ms step to +200 mV, followed by repolarization to voltages from +80 to −200 mV (Fig. 4). In the absence of Y3+, the “tail” current through T channels decays by a combination of inactivation (O → I) and deactivation (O → C) (Serrano et al., 1999). At depolarized voltages (+80 mV and −40 mV in Fig. 4 A), channels inactivate in a nearly voltage-independent manner (Fig. 4 B, open squares). At more negative voltages, deactivation dominates (−160 mV in Fig. 4 A). T-channel deactivation depends almost exponentially on voltage (Herrington and Lingle, 1992; Serrano et al., 1999), illustrated by a nearly linear relation between log(τ) and voltage from −60 to −200 mV (Fig. 4 B, open squares). The tail current τ's following steps to +200 mV (Fig. 4 B) agree well with values measured previously following steps to +60 mV (Serrano et al., 1999), suggesting that brief steps to +200 mV do not affect channel gating in unexpected ways.


Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel.

Obejero-Paz CA, Gray IP, Jones SW - J. Gen. Physiol. (2004)

Reentry of Y3+ into open channels. (A) Effect of 1 μM Y3+ on tail currents. A 1-ms step to +200 mV was used to both activate channels and relieve Y3+ block. Currents are shown following repolarization to +80 mV, −40 mV, and −160 mV. Currents at +200 mV are off scale. 1.5 kHz Gaussian filter. (B) Acceleration of tail currents by Y3+. Tail currents were fitted by single exponentials in control conditions, and in four concentrations of Y3+. Values are not shown at +20 mV, near the reversal potential. n = 4–6. (C) Voltage- and concentration-dependent reblocking. For each cell, at each voltage, the blocking rate was calculated from tail current time constants in Y3+ and in control conditions (Eq. 1).
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fig4: Reentry of Y3+ into open channels. (A) Effect of 1 μM Y3+ on tail currents. A 1-ms step to +200 mV was used to both activate channels and relieve Y3+ block. Currents are shown following repolarization to +80 mV, −40 mV, and −160 mV. Currents at +200 mV are off scale. 1.5 kHz Gaussian filter. (B) Acceleration of tail currents by Y3+. Tail currents were fitted by single exponentials in control conditions, and in four concentrations of Y3+. Values are not shown at +20 mV, near the reversal potential. n = 4–6. (C) Voltage- and concentration-dependent reblocking. For each cell, at each voltage, the blocking rate was calculated from tail current time constants in Y3+ and in control conditions (Eq. 1).
Mentions: To examine reblocking by Y3+, channels were first unblocked by a 1-ms step to +200 mV, followed by repolarization to voltages from +80 to −200 mV (Fig. 4). In the absence of Y3+, the “tail” current through T channels decays by a combination of inactivation (O → I) and deactivation (O → C) (Serrano et al., 1999). At depolarized voltages (+80 mV and −40 mV in Fig. 4 A), channels inactivate in a nearly voltage-independent manner (Fig. 4 B, open squares). At more negative voltages, deactivation dominates (−160 mV in Fig. 4 A). T-channel deactivation depends almost exponentially on voltage (Herrington and Lingle, 1992; Serrano et al., 1999), illustrated by a nearly linear relation between log(τ) and voltage from −60 to −200 mV (Fig. 4 B, open squares). The tail current τ's following steps to +200 mV (Fig. 4 B) agree well with values measured previously following steps to +60 mV (Serrano et al., 1999), suggesting that brief steps to +200 mV do not affect channel gating in unexpected ways.

Bottom Line: Closed channels were also blocked by Y(3+) at a concentration-dependent rate, only eightfold slower than open-channel block.When extracellular Ca(2+) was replaced with Ba(2+), the rate of open block by Y(3+) was unaffected, but closed block was threefold faster than in Ca(2+), suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate.Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular "pore gate" has also been proposed. We have used the voltage dependence of block by extracellular Y(3+) as a tool to locate the activation gate of the alpha1G (Ca(V)3.1) T-type calcium channel. Y(3+) block exhibited no clear voltage dependence from -40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y(3+) at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca(2+) was replaced with Ba(2+), the rate of open block by Y(3+) was unaffected, but closed block was threefold faster than in Ca(2+), suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.

Show MeSH
Related in: MedlinePlus