scholarly journals Mechanism of Tetracaine Block of Cyclic Nucleotide-gated Channels

1997 ◽  
Vol 109 (1) ◽  
pp. 3-14 ◽  
Author(s):  
Anthony A. Fodor ◽  
Sharona E. Gordon ◽  
William N. Zagotta
Keyword(s):  
Conformational Changes ◽  
Cyclic Nucleotide ◽  
Channel Blockers ◽  
Response Curves ◽  
Apparent Affinity ◽  
Ion Channel Blockers ◽  
Cyclic Nucleotide Gated Channels ◽  
Closed State ◽  
State Dependent ◽  
Voltage Dependent

Local anesthetics are a diverse group of ion channel blockers that can be used to probe conformational changes in the pore. We examined the effects of the local anesthetic tetracaine on rod and olfactory cyclic nucleotide-gated channels expressed from subunit 1 in Xenopus oocytes. We found that 40 μM tetracaine effectively blocked the bovine rod channel but not the rat olfactory channel at saturating concentrations of cGMP. By testing chimeric channels containing regions of sequence from both rod and olfactory channels, we found that determinants of apparent affinity for tetracaine at saturating cGMP did not map to any one region of the channel sequence. Rather, the differences in apparent affinity could be explained by differences between the chimeras in the free energy of the opening allosteric transition. If a channel construct (such as the rod channel) spent appreciable time in the closed state at saturating cGMP, then it had a high apparent affinity for tetracaine. If, on the other hand, a channel construct (such as the olfactory channel) spent little time in the closed state at saturating cGMP, then it had a low apparent affinity for tetracaine. Furthermore, tetracaine became more effective at low concentrations of cGMP and at saturating concentrations of cAMP, conditions which permit the channels to spend more time in the closed configuration. These results were well fit by a model in which tetracaine binds more tightly to the closed channel than to the open channel. Dose-response curves for tetracaine in the presence of saturating cGMP are well fit with a Michaelis-Menten binding scheme Indicating that a single tetracaine molecule is sufficient to produce block. In addition, tetracaine block is voltage dependent with an effective zδ of +0.56. These data are consistent with a pore-block hypothesis. The finding that tetracaine is a state-dependent pore blocker suggests that the inner mouth of the pore of cyclic nucleotide-gated channels undergoes a conformational change during channel opening.

scholarly journals Binding of benzocaine in batrachotoxin-modified Na+ channels. State-dependent interactions.

1994 ◽  
Vol 103 (3) ◽  
pp. 501-518 ◽  
Author(s):  
G K Wang ◽  
S Y Wang
Keyword(s):  
Binding Affinity ◽  
Open Channel ◽  
Gh3 Cells ◽  
Na Channel ◽  
Channel Blockers ◽  
Na Channels ◽  
Response Curves ◽  
State Dependent ◽  
Voltage Dependent ◽  
Command Signals

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX-modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use-dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.

Expression of dihydropyridine resistance differs in porcine bronchial and tracheal smooth muscle

1994 ◽  
Vol 267 (2) ◽  
pp. L106-L112 ◽  
Author(s):  
T. L. Croxton ◽  
C. Fleming ◽  
C. A. Hirshman
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Isometric Tension ◽  
Tracheal Smooth Muscle ◽  
Airway Smooth Muscle Cells ◽  
Channel Blockers ◽  
Response Curves ◽  
Voltage Dependent ◽  
Relaxation Responses ◽  
Ca2 Channels

Voltage-dependent and receptor-operated Ca2+ entry mechanisms have been demonstrated in airway smooth muscle, but their relative importance for maintenance of contraction is unknown. Blockade of voltage-dependent Ca2+ channels (VDC) has produced inconsistent relaxation. We postulated regional variations in Ca2+ handling by airway smooth muscle cells and compared the efficacy of dihydropyridine VDC blockers in tracheas and bronchi. Porcine tracheal smooth muscle strips and bronchial rings were mounted in tissue baths filled with physiological solutions and isometric tension was measured. Tissues were precontracted with carbachol or KCl, and relaxation dose-response curves to nifedipine, Mn2+, or Cd2+ were obtained. Relaxation responses to nifedipine were significantly different in carbachol-contracted tracheas and bronchi. Whereas carbachol-contracted tracheal muscle completely relaxed with 10(-6) M nifedipine, bronchial smooth muscle relaxed < 50%. In contrast, KCl-contracted bronchial muscle was completely relaxed by nifedipine. The nonspecific Ca2+ channel blockers Mn2+ and Cd2+ produced similar relaxation responses in each tissue. Thus VDC are the predominant mechanism for Ca2+ entry in porcine tracheal smooth muscle, but a dihydropyridine-insensitive pathway is functionally important in carbachol-contracted porcine bronchi. Regional variation may account for apparent inconsistencies between previous studies.

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 Conformational Changes in the Pore of CLC-0

2003 ◽  
Vol 122 (3) ◽  
pp. 277-294 ◽  
Author(s):  
Alessio Accardi ◽  
Michael Pusch
Keyword(s):  
Conformational Changes ◽  
Single Channel ◽  
General Structure ◽  
Point Mutations ◽  
Clofibric Acid ◽  
Selectivity Filter ◽  
Side Chain ◽  
Closed State ◽  
State Dependent ◽  
Channel Opening

The Torpedo Cl− channel, CLC-0, is inhibited by clofibric acid derivatives from the intracellular side. We used the slow gate-deficient mutant CLC-0C212S to investigate the mechanism of block by the clofibric acid–derivative p-chlorophenoxy-acetic acid (CPA). CPA blocks open channels with low affinity (KDO= 45 mM at 0 mV) and shows fast dissociation (koff = 490 s−1 at −140 mV). In contrast, the blocker binds to closed channels with higher affinity and with much slower kinetics. This state-dependent block coupled with the voltage dependence of the gating transitions results in a highly voltage-dependent inhibition of macroscopic currents (KD ∼1 mM at −140 mV; KD ∼65 mM at 60 mV). The large difference in CPA affinity of the open and closed state suggests that channel opening involves more than just a local conformational rearrangement. On the other hand, in a recent work (Dutzler, R., E.B. Campbell, and R. MacKinnon. 2003. Science. 300:108–112) it was proposed that the conformational change underlying channel opening is limited to a movement of a single side chain. A prediction of this latter model is that mutations that influence CPA binding to the channel should affect the affinities for an open and closed channel in a similar manner since the general structure of the pore remains largely unchanged. To test this hypothesis we introduced point mutations in four residues (S123, T471, Y512, and K519) that lie close to the intracellular pore mouth or to the putative selectivity filter. Mutation T471S alters CPA binding exclusively to closed channels. Pronounced effects on the open channel block are observed in three other mutants, S123T, Y512A, and K519Q. Together, these results collectively suggest that the structure of the CPA binding site is different in the open and closed state. Finally, replacement of Tyr 512, a residue directly coordinating the central Cl− ion in the crystal structure, with Phe or Ala has very little effect on single channel conductance and selectivity. These observations suggest that channel opening in CLC-0 consists in more than a movement of a side chain and that other parts of the channel and of the selectivity filter are probably involved.

scholarly journals Access of Quaternary Ammonium Blockers to the Internal Pore of Cyclic Nucleotide-gated Channels: Implications for the Location of the Gate

2006 ◽  
Vol 127 (5) ◽  
pp. 481-494 ◽  
Author(s):  
Jorge E. Contreras ◽  
Miguel Holmgren
Keyword(s):  
Binding Site ◽  
Quaternary Ammonium ◽  
Cyclic Nucleotide ◽  
Single Channel ◽  
Dependent Manner ◽  
Receptor Binding Site ◽  
Α Subunit ◽  
Cyclic Nucleotide Gated Channels ◽  
Closed State ◽  
Kv Channels

Cyclic nucleotide-gated (CNG) channels play important roles in the transduction of visual and olfactory information by sensing changes in the intracellular concentration of cyclic nucleotides. We have investigated the interactions between intracellularly applied quaternary ammonium (QA) ions and the α subunit of rod cyclic nucleotide-gated channels. We have used a family of alkyl-triethylammonium derivatives in which the length of one chain is altered. These QA derivatives blocked the permeation pathway of CNG channels in a concentration- and voltage-dependent manner. For QA compounds with tails longer than six methylene groups, increasing the length of the chain resulted in higher apparent affinities of ∼1.2 RT per methylene group added, which is consistent with the presence of a hydrophobic pocket within the intracellular mouth of the channel that serves as part of the receptor binding site. At the single channel level, decyltriethyl ammonium (C10-TEA) ions did not change the unitary conductance but they did reduce the apparent mean open time, suggesting that the blocker binds to open channels. We provide four lines of evidence suggesting that QA ions can also bind to closed channels: (1) the extent of C10-TEA blockade at subsaturating [cGMP] was larger than at saturating agonist concentration, (2) under saturating concentrations of cGMP, cIMP, or cAMP, blockade levels were inversely correlated with the maximal probability of opening achieved by each agonist, (3) in the closed state, MTS reagents of comparable sizes to QA ions were able to modify V391C in the inner vestibule of the channel, and (4) in the closed state, C10-TEA was able to slow the Cd2+ inhibition observed in V391C channels. These results are in stark contrast to the well-established QA blockade mechanism in Kv channels, where these compounds can only access the inner vestibule in the open state because the gate that opens and closes the channel is located cytoplasmically with respect to the binding site of QA ions. Therefore, in the context of Kv channels, our observations suggest that the regions involved in opening and closing the permeation pathways in these two types of channels are different.

scholarly journals Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel

2012 ◽  
Vol 140 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Shengjun Wu ◽  
Weihua Gao ◽  
Changan Xie ◽  
Xinping Xu ◽  
Christina Vorvis ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Cyclic Nucleotide ◽  
Binding Domain ◽  
Hcn Channels ◽  
Channel Blockers ◽  
Structural Elements ◽  
Hcn Channel ◽  
Channel Interaction ◽  
State Dependent ◽  
Activation Gate

Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide–gated (CNG) and hyperpolarization-activated, cyclic nucleotide–regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand–channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand–whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs+ and Mg2+, effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.

scholarly journals Opening Mechanism of a Cyclic Nucleotide–gated Channel Based on Analysis of Single Channels Locked in Each Liganded State

1999 ◽  
Vol 113 (6) ◽  
pp. 873-895 ◽  
Author(s):  
MariaLuisa Ruiz ◽  
Jeffrey W. Karpen
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Cyclic Nucleotide ◽  
Single Channel ◽  
Kinetic Mechanism ◽  
Fixed Number ◽  
Single Channels ◽  
Cyclic Nucleotide Gated Channels ◽  
Gated Channel ◽  
Conductance States

Cyclic nucleotide–gated channels contain four subunits, each with a binding site for cGMP or cAMP in the cytoplasmic COOH-terminal domain. Previous studies of the kinetic mechanism of activation have been hampered by the complication that ligands are continuously binding and unbinding at each of these sites. Thus, even at the single channel level, it has been difficult to distinguish changes in behavior that arise from a channel with a fixed number of ligands bound from those that occur upon the binding and unbinding of ligands. For example, it is often assumed that complex behaviors like multiple conductance levels and bursting occur only as a consequence of changes in the number of bound ligands. We have overcome these ambiguities by covalently tethering one ligand at a time to single rod cyclic nucleotide–gated channels (Ruiz, ML., and J.W. Karpen. 1997. Nature. 389:389–392). We find that with a fixed number of ligands locked in place the channel freely moves between three conductance states and undergoes bursting behavior. Furthermore, a thorough kinetic analysis of channels locked in doubly, triply, and fully liganded states reveals more than one kinetically distinguishable state at each conductance level. Thus, even when the channel contains a fixed number of bound ligands, it can assume at least nine distinct states. Such complex behavior is inconsistent with simple concerted or sequential allosteric models. The data at each level of liganding can be successfully described by the same connected state model (with different rate constants), suggesting that the channel undergoes the same set of conformational changes regardless of the number of bound ligands. A general allosteric model, which postulates one conformational change per subunit in both the absence and presence of ligand, comes close to providing enough kinetically distinct states. We propose an extension of this model, in which more than one conformational change per subunit can occur during the process of channel activation.

scholarly journals Gating of Cyclic Nucleotide Gated Channels is also Voltage Dependent

2012 ◽  
Vol 102 (3) ◽  
pp. 130a ◽  
Author(s):  
Arin Marchesi ◽  
Monica Mazzolini ◽  
Vincent Torre
Keyword(s):  
Cyclic Nucleotide ◽  
Cyclic Nucleotide Gated Channels ◽  
Voltage Dependent

scholarly journals Closed state-coupled C-type inactivation in BK channels

2016 ◽  
Vol 113 (25) ◽  
pp. 6991-6996 ◽  
Author(s):  
Jiusheng Yan ◽  
Qin Li ◽  
Richard W. Aldrich
Keyword(s):  
Conformational Changes ◽  
Bk Channels ◽  
Bk Channel ◽  
Membrane Potentials ◽  
Selectivity Filter ◽  
Open Probability ◽  
Closed State ◽  
State Dependent ◽  
Channel Opening ◽  
Activation Gate

Ion channels regulate ion flow by opening and closing their pore gates. K+ channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We hypothesized that the BK channel’s activation gate may spatially overlap or coexist with the C-type inactivation gate at or near the selectivity filter. We induced C-type inactivation in BK channels and studied the relationship between activation/deactivation and C-type inactivation/recovery. We observed prominent slow C-type inactivation/recovery in BK channels by an extreme low concentration of extracellular K+ together with a Y294E/K/Q/S or Y279F mutation whose equivalent in Shaker channels (T449E/K/D/Q/S or W434F) caused a greatly accelerated rate of C-type inactivation or constitutive C-inactivation. C-type inactivation in most K+ channels occurs upon sustained membrane depolarization or channel opening and then recovers during hyperpolarized membrane potentials or channel closure. However, we found that the BK channel C-type inactivation occurred during hyperpolarized membrane potentials or with decreased intracellular calcium ([Ca2+]i) and recovered with depolarized membrane potentials or elevated [Ca2+]i. Constitutively open mutation prevented BK channels from C-type inactivation. We concluded that BK channel C-type inactivation is closed state-dependent and that its extents and rates inversely correlate with channel-open probability. Because C-type inactivation can involve multiple conformational changes at the selectivity filter, we propose that the BK channel’s normal closing may represent an early conformational stage of C-type inactivation.

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