scholarly journals cAMP binds to closed, inactivated, and open sea urchin HCN channels in a state-dependent manner

2018 ◽  
Vol 151 (2) ◽  
pp. 200-213 ◽  
Author(s):  
Vinay Idikuda ◽  
Weihua Gao ◽  
Zhuocheng Su ◽  
Qinglian Liu ◽  
Lei Zhou
Keyword(s):  
Sea Urchin ◽  
Transmembrane Domain ◽  
Single Point ◽  
Single Point Mutation ◽  
Fluorescence Signal ◽  
Hcn Channels ◽  
Dependent Manner ◽  
State Dependent ◽  
Dependent Inactivation ◽  
Voltage Dependent

Hyperpolarization-activated cyclic-nucleotide–modulated (HCN) channels are nonselective cation channels that regulate electrical activity in the heart and brain. Previous studies of mouse HCN2 (mHCN2) channels have shown that cAMP binds preferentially to and stabilizes these channels in the open state—a simple but elegant implementation of ligand-dependent gating. Distinct from mammalian isoforms, the sea urchin (spHCN) channel exhibits strong voltage-dependent inactivation in the absence of cAMP. Here, using fluorescently labeled cAMP molecules as a marker for cAMP binding, we report that the inactivated spHCN channel displays reduced cAMP binding compared with the closed channel. The reduction in cAMP binding is a voltage-dependent process but proceeds at a much slower rate than the movement of the voltage sensor. A single point mutation in the last transmembrane domain near the channel’s gate, F459L, abolishes inactivation and concurrently reverses the response to hyperpolarizing voltage steps from a decrease to an increase in cAMP binding. ZD7288, an open channel blocker that interacts with a region close to the activation/inactivation gate, dampens the reduction of cAMP binding to inactivated spHCN channels. In addition, compared with closed and “locked” closed channels, increased cAMP binding is observed in channels purposely locked in the open state upon hyperpolarization. Thus, the order of cAMP-binding affinity, measured by the fluorescence signal from labeled cAMP, ranges from high in the open state to intermediate in the closed state to low in the inactivated state. Our work on spHCN channels demonstrates intricate state-dependent communications between the gate and ligand-binding domain and provides new mechanistic insight into channel inactivation/desensitization.

scholarly journals S4 Movement in a Mammalian HCN Channel

2003 ◽  
Vol 123 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Sriharsha Vemana ◽  
Shilpi Pandey ◽  
H. Peter Larsson
Keyword(s):  
Voltage Sensor ◽  
K Channel ◽  
Hcn Channels ◽  
Dependent Manner ◽  
Hcn Channel ◽  
Voltage Range ◽  
Kv Channels ◽  
State Dependent ◽  
Voltage Dependent ◽  
Hcn1 Channels

Hyperpolarization-activated, cyclic nucleotide–gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal–deleted spHCN. This channel appeared to be similar to a COOH-terminal–deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels.

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
Author(s):  
Kevin J. Gingrich ◽  
Son Tran ◽  
Igor M. Nikonorov ◽  
Thomas J. Blanck
Keyword(s):  
Charge Transfer ◽  
Calcium Channels ◽  
Dependent Manner ◽  
Current Time ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

Inhibition by Imipramine of the Voltage-Dependent K+ Channel in Rabbit Coronary Arterial Smooth Muscle Cells

2020 ◽  
Vol 178 (2) ◽  
pp. 302-310
Author(s):  
Jin Ryeol An ◽  
Mi Seon Seo ◽  
Hee Seok Jung ◽  
Ryeon Heo ◽  
Minji Kang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Dependent Manner ◽  
Arterial Smooth Muscle ◽  
Closed State ◽  
Kv Channels ◽  
State Dependent ◽  
Voltage Dependent ◽  
Arterial Smooth Muscle Cells

Abstract Imipramine, a tricyclic antidepressant, is used in the treatment of depressive disorders. However, the effect of imipramine on vascular ion channels is unclear. Therefore, using a patch-clamp technique we examined the effect of imipramine on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells. Kv channels were inhibited by imipramine in a concentration-dependent manner, with an IC50 value of 5.55 ± 1.24 µM and a Hill coefficient of 0.73 ± 0.1. Application of imipramine shifted the steady-state activation curve in the positive direction, indicating that imipramine-induced inhibition of Kv channels was mediated by influencing the voltage sensors of the channels. The recovery time constants from Kv-channel inactivation were increased in the presence of imipramine. Furthermore, the application of train pulses (of 1 or 2 Hz) progressively augmented the imipramine-induced inhibition of Kv channels, suggesting that the inhibitory effect of imipramine is use (state) dependent. The magnitude of Kv current inhibition by imipramine was similar during the first, second, and third depolarizing pulses. These results indicate that imipramine-induced inhibition of Kv channels mainly occurs in the closed state. The imipramine-mediated inhibition of Kv channels was associated with the Kv1.5 channel, not the Kv2.1 or Kv7 channel. Inhibition of Kv channels by imipramine caused vasoconstriction. From these results, we conclude that imipramine inhibits vascular Kv channels in a concentration- and use (closed-state)-dependent manner by changing their gating properties regardless of its own function.

scholarly journals Inactivation of N-type calcium current in chick sensory neurons: calcium and voltage dependence.

1994 ◽  
Vol 104 (2) ◽  
pp. 311-336 ◽  
Author(s):  
D H Cox ◽  
K Dunlap
Keyword(s):  
Voltage Dependence ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Slow Inactivation ◽  
Fast Inactivation ◽  
Drg Neurons ◽  
Rapid Phase ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Dependent Processes

We have studied the inactivation of high-voltage-activated (HVA), omega-conotoxin-sensitive, N-type Ca2+ current in embryonic chick dorsal root ganglion (DRG) neurons. Voltage steps from -80 to 0 mV produced inward Ca2+ currents that inactivated in a biphasic manner and were fit well with the sum of two exponentials (with time constants of approximately 100 ms and > 1 s). As reported previously, upon depolarization of the holding potential to -40 mV, N current amplitude was significantly reduced and the rapid phase of inactivation all but eliminated (Nowycky, M. C., A. P. Fox, and R. W. Tsien. 1985. Nature. 316:440-443; Fox, A. P., M. C. Nowycky, and R. W. Tsien. 1987a. Journal of Physiology. 394:149-172; Swandulla, D., and C. M. Armstrong. 1988. Journal of General Physiology. 92:197-218; Plummer, M. R., D. E. Logothetis, and P. Hess. 1989. Neuron. 2:1453-1463; Regan, L. J., D. W. Sah, and B. P. Bean. 1991. Neuron. 6:269-280; Cox, D. H., and K. Dunlap. 1992. Journal of Neuroscience. 12:906-914). Such kinetic properties might be explained by a model in which N channels inactivate by both fast and slow voltage-dependent processes. Alternatively, kinetic models of Ca-dependent inactivation suggest that the biphasic kinetics and holding-potential-dependence of N current inactivation could be due to a combination of Ca-dependent and slow voltage-dependent inactivation mechanisms. To distinguish between these possibilities we have performed several experiments to test for the presence of Ca-dependent inactivation. Three lines of evidence suggest that N channels inactivate in a Ca-dependent manner. (a) The total extent of inactivation increased 50%, and the ratio of rapid to slow inactivation increased approximately twofold when the concentration of the Ca2+ buffer, EGTA, in the patch pipette was reduced from 10 to 0.1 mM. (b) With low intracellular EGTA concentrations (0.1 mM), the ratio of rapid to slow inactivation was additionally increased when the extracellular Ca2+ concentration was raised from 0.5 to 5 mM. (c) Substituting Na+ for Ca2+ as the permeant ion eliminated the rapid phase of inactivation. Other results do not support the notion of current-dependent inactivation, however. Although high intracellular EGTA (10 mM) or BAPTA (5 mM) concentrations suppressed the rapid phase inactivation, they did not eliminate it. Increasing the extracellular Ca2+ from 0.5 to 5 mM had little effect on this residual fast inactivation, indicating that it is not appreciably sensitive to Ca2+ influx under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Protriptyline, a tricyclic antidepressant, inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

2020 ◽  
Vol 52 (3) ◽  
pp. 320-327 ◽  
Author(s):  
Jin Ryeol An ◽  
Hojung Kang ◽  
Hongliang Li ◽  
Mi Seon Seo ◽  
Hee Seok Jung ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Tricyclic Antidepressant ◽  
Dependent Manner ◽  
Kv Channels ◽  
State Dependent ◽  
Voltage Dependent ◽  
Arterial Smooth Muscle Cells

Abstract In this study, we explore the inhibitory effects of protriptyline, a tricyclic antidepressant drug, on voltage-dependent K+ (Kv) channels of rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Protriptyline inhibited the vascular Kv current in a concentration-dependent manner, with an IC50 value of 5.05 ± 0.97 μM and a Hill coefficient of 0.73 ± 0.04. Protriptyline did not affect the steady-state activation kinetics. However, the drug shifted the steady-state inactivation curve to the left, suggesting that protriptyline inhibited the Kv channels by changing their voltage sensitivity. Application of 20 repetitive train pulses (1 or 2 Hz) progressively increased the protriptyline-induced inhibition of the Kv current, suggesting that protriptyline inhibited Kv channels in a use (state)-dependent manner. The extent of Kv current inhibition by protriptyline was similar during the first, second, and third step pulses. These results suggest that protriptyline-induced inhibition of the Kv current mainly occurs principally in the closed state. The increase in the inactivation recovery time constant in the presence of protriptyline also supported use (state)-dependent inhibition of Kv channels by the drug. In the presence of the Kv1.5 inhibitor, protriptyline did not induce further inhibition of the Kv channels. However, pretreatment with a Kv2.1 or Kv7 inhibitor induced further inhibition of Kv current to a similar extent to that observed with protriptyline alone. Thus, we conclude that protriptyline inhibits the vascular Kv channels in a concentration- and use-dependent manner by changing their gating properties. Furthermore, protriptyline-induced inhibition of Kv channels mainly involves the Kv1.5.

Coexpression with β1-subunit modifies the kinetics and fatty acid block of hH1α Na+channels

2000 ◽  
Vol 279 (1) ◽  
pp. H35-H46 ◽  
Author(s):  
Yong-Fu Xiao ◽  
Sterling N. Wright ◽  
Ging Kuo Wang ◽  
James P. Morgan ◽  
Alexander Leaf
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Monounsaturated Fatty Acids ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Α Subunit ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Steady State Inactivation

Voltage-gated cardiac Na+ channels are composed of α- and β1-subunits. In this study β1-subunit was cotransfected with the α-subunit of the human cardiac Na+ channel (hH1α) in human embryonic kidney (HEK293t) cells. The effects of this coexpression on the kinetics and fatty acid-induced suppression of Na+currents were assessed. Current density was significantly greater in HEK293t cells coexpressing α- and β1-subunits ( I Na,αβ) than in HEK293t cells expressing α-subunit alone ( I Na,α). Compared with I Na,α, the voltage-dependent inactivation and activation of I Na,αβ were significantly shifted in the depolarizing direction. In addition, coexpression with β1-subunit prolonged the duration of recovery from inactivation. Eicosapentaenoic acid [EPA, C20:5(n–3)] significantly reduced I Na,αβ in a concentration-dependent manner and at 5 μM shifted the midpoint voltage of the steady-state inactivation by −22 ± 1 mV. EPA also significantly accelerated channel transition from the resting state to the inactivated state and prolonged the recovery time from inactivation. Docosahexaenoic acid [C22:6(n–3)], α-linolenic acid [C18:3(n–3)], and conjugated linoleic acid [C18:2(n–6)] at 5 μM significantly inhibited both I Na,αβ and I Na,α.In contrast, saturated and monounsaturated fatty acids had no effects on I Na,αβ. This finding differs from the results for I Na,α, which was significantly inhibited by both saturated and unsaturated fatty acids. Our data demonstrate that functional association of β1-subunit with hH1α modifies the kinetics and fatty acid block of the Na+ channel.

Direct Inhibition of Ih by Analgesic Loperamide in Rat DRG Neurons

2007 ◽  
Vol 97 (5) ◽  
pp. 3713-3721 ◽  
Author(s):  
Dmitry V. Vasilyev ◽  
Qin Shan ◽  
Yan Lee ◽  
Scott C. Mayer ◽  
Mark R. Bowlby ◽  
...  
Keyword(s):  
Steady State ◽  
Opioid Receptors ◽  
Small Diameter ◽  
Hcn Channels ◽  
Voltage Sensitivity ◽  
Pain Processing ◽  
Dependent Manner ◽  
Drg Neurons ◽  
Extracellular Region ◽  
Voltage Dependent

Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels are responsible for the functional hyperpolarization-activated current ( Ih) in dorsal root ganglion (DRG) neurons, playing an important role in pain processing. We found that the known analgesic loperamide inhibited Ih channels in rat DRG neurons. Loperamide blocked Ih in a concentration-dependent manner, with an IC50 = 4.9 ± 0.6 and 11.0 ± 0.5 μM for large- and small-diameter neurons, respectively. Loperamide-induced Ih inhibition was unrelated to the activation of opioid receptors and was reversible, voltage-dependent, use-independent, and was associated with a negative shift of V1/2 for Ih steady-state activation. Loperamide block of Ih was voltage-dependent, gradually decreasing at more hyperpolarized membrane voltages from 89% at –60 mV to 4% at –120 mV in the presence of 3.7 μM loperamide. The voltage sensitivity of block can be explained by a loperamide-induced shift in the steady-state activation of Ih. Inclusion of 10 μM loperamide into the recording pipette did not affect Ih voltage for half-maximal activation, activation kinetics, and the peak current amplitude, whereas concurrent application of equimolar external loperamide produced a rapid, reversible Ih inhibition. The observed loperamide-induced Ih inhibition was not caused by the activation of peripheral opioid receptors because the broad-spectrum opioid receptor antagonist naloxone did not reverse Ih inhibition. Therefore we suggest that loperamide inhibits Ih by direct binding to the extracellular region of the channel. Because Ih channels are involved in pain processing, loperamide-induced inhibition of Ih channels could provide an additional molecular mechanism for its analgesic action.

scholarly journals RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells

2019 ◽  
Vol 472 (1) ◽  
pp. 3-25 ◽  
Author(s):  
Nadine J. Ortner ◽  
Alexandra Pinggera ◽  
Nadja T. Hofer ◽  
Anita Siller ◽  
Niels Brandt ◽  
...  
Keyword(s):  
Hair Cells ◽  
Splice Variant ◽  
Glutamate Release ◽  
Inactivation Kinetics ◽  
Dependent Manner ◽  
Voltage Range ◽  
Inner Hair Cells ◽  
Whole Cell Patch Clamp ◽  
Dependent Inactivation ◽  
Voltage Dependent

AbstractCav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3L) and a short splice variant (Cav1.342A) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary β3 subunit, RIM2α alone (Cav1.342A) or RIM2α/RBP2 (Cav1.3L) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored β2 variants (β2a, β2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel’s activation threshold toward more hyperpolarized potentials. Taken together, the association with “slow” β subunits (β2a, β2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function.

scholarly journals Voltage-Dependent Inactivation of MscS Occurs Independently of the Positively Charged Residues in the Transmembrane Domain

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Takeshi Nomura ◽  
Masahiro Sokabe ◽  
Kenjiro Yoshimura
Keyword(s):  
Voltage Dependence ◽  
Transmembrane Domain ◽  
Wild Type ◽  
Dependent Inactivation ◽  
Mechanical Threshold ◽  
Voltage Dependent ◽  
Arginine Residues ◽  
Tm Domain ◽  
Small Conductance ◽  
Positively Charged

MscS (mechanosensitive channel of small conductance) is ubiquitously found among bacteria and plays a major role in avoiding cell lysis upon rapid osmotic downshock. The gating of MscS is modulated by voltage, but little is known about how MscS senses membrane potential. Three arginine residues (Arg-46, Arg-54, and Arg-74) in the transmembrane (TM) domain are possible to respond to voltage judging from the MscS structure. To examine whether these residues are involved in the voltage dependence of MscS, we neutralized the charge of each residue by substituting with asparagine (R46N, R54N, and R74N). Mechanical threshold for the opening of the expressed wild-type MscS and asparagine mutants did not change with voltage in the range from-40 to +100 mV. By contrast, inactivation process of wild-type MscS was strongly affected by voltage. The wild-type MscS inactivated at +60 to +80 mV but not at-60 to +40 mV. The voltage dependence of the inactivation rate of all mutants tested, that is, R46N, R54N, R74N, and R46N/R74N MscS, was almost indistinguishable from that of the wild-type MscS. These findings indicate that the voltage dependence of the inactivation occurs independently of the positive charges of R46, R54, and R74.

scholarly journals State-Dependent Inactivation of the α1g T-Type Calcium Channel

1999 ◽  
Vol 114 (2) ◽  
pp. 185-202 ◽  
Author(s):  
Jose R. Serrano ◽  
Edward Perez-Reyes ◽  
Stephen W. Jones
Keyword(s):  
Kinetic Model ◽  
Hek 293 ◽  
Voltage Sensors ◽  
State Dependent ◽  
State Recovery ◽  
Recovery From Inactivation ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Kinetics Of

We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the α1G channel, with symmetrical Na+i and Na+o and 2 mM Ca2+o. After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential −100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from −120 to −70 mV \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{e-fold\;for}}\;31\;{\mathrm{mV}};\;{\mathrm{{\tau}}}\;=\;2.5\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document}, but \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}{\mathrm{{\tau}}}\;=\;12{\raisebox{1mm}{\line(1,0){6}}}17\;{\mathrm{ms\;from}}-40\;{\mathrm{to}}\;+60\;{\mathrm{mV}}\end{equation*}\end{document}. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100–300 ms, inactivation was strong but incomplete (∼98%). Inactivation was also produced by long, weak depolarizations \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{{\tau}}}\;=\;220\;{\mathrm{ms\;at}}\;-80\;{\mathrm{mV}};\;{\mathrm{V}}_{1/2}\;=\;-82\;{\mathrm{mV}})\end{equation*}\end{document}, which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{{\tau}}}\;=\;85\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document}, but weakly voltage dependent. Recovery was similar after 60-ms steps to −20 mV or 600-ms steps to −70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at −100 mV during recovery from inactivation, consistent with ≤8% of the channels recovering through the open state. The results are well described by a kinetic model where inactivation is allosterically coupled to the movement of the first three voltage sensors to activate. One consequence of state-dependent inactivation is that α1G channels continue to inactivate after repolarization, primarily from the open state, which leads to cumulative inactivation during repetitive pulses.

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