scholarly journals Scanning the Intracellular S6 Activation Gate in the Shaker K+ Channel

2002 ◽  
Vol 119 (6) ◽  
pp. 521-531 ◽  
Author(s):  
David H. Hackos ◽  
Tsg-Hui Chang ◽  
Kenton J. Swartz
Keyword(s):  
Ionic Currents ◽  
K Channel ◽  
Dependent Manner ◽  
Kv Channel ◽  
Closed State ◽  
Kv Channels ◽  
Gate Structure ◽  
Activation Gate ◽  
Voltage Dependent ◽  
Gate Region

In Kv channels, an activation gate is thought to be located near the intracellular entrance to the ion conduction pore. Although the COOH terminus of the S6 segment has been implicated in forming the gate structure, the residues positioned at the occluding part of the gate remain undetermined. We use a mutagenic scanning approach in the Shaker Kv channel, mutating each residue in the S6 gate region (T469-Y485) to alanine, tryptophan, and aspartate to identify positions that are insensitive to mutation and to find mutants that disrupt the gate. Most mutants open in a steeply voltage-dependent manner and close effectively at negative voltages, indicating that the gate structure can both support ion flux when open and prevent it when closed. We find several mutant channels where macroscopic ionic currents are either very small or undetectable, and one mutant that displays constitutive currents at negative voltages. Collective examination of the three types of substitutions support the notion that the intracellular portion of S6 forms an activation gate and identifies V478 and F481 as candidates for occlusion of the pore in the closed state.

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 Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K+ channel

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Anirban Banerjee ◽  
Alice Lee ◽  
Ernest Campbell ◽  
Roderick MacKinnon
Keyword(s):  
Electron Density ◽  
Selectivity Filter ◽  
K Channel ◽  
Wild Type ◽  
Kv Channel ◽  
Pore Blocking ◽  
Kv Channels ◽  
Filter Structure ◽  
Conduction Pathway ◽  
Voltage Dependent

Pore-blocking toxins inhibit voltage-dependent K+ channels (Kv channels) by plugging the ion-conduction pathway. We have solved the crystal structure of paddle chimera, a Kv channel in complex with charybdotoxin (CTX), a pore-blocking toxin. The toxin binds to the extracellular pore entryway without producing discernable alteration of the selectivity filter structure and is oriented to project its Lys27 into the pore. The most extracellular K+ binding site (S1) is devoid of K+ electron-density when wild-type CTX is bound, but K+ density is present to some extent in a Lys27Met mutant. In crystals with Cs+ replacing K+, S1 electron-density is present even in the presence of Lys27, a finding compatible with the differential effects of Cs+ vs K+ on CTX affinity for the channel. Together, these results show that CTX binds to a K+ channel in a lock and key manner and interacts directly with conducting ions inside the selectivity filter.

The Voltage-Dependent K+ Channel Family

2019 ◽  
Author(s):  
Hanne B. Rasmussen ◽  
James S. Trimmer
Keyword(s):  
Ion Channels ◽  
Neuronal Excitability ◽  
Expression Patterns ◽  
Basic Knowledge ◽  
K Channel ◽  
Dependent Manner ◽  
Kv Channels ◽  
Voltage Dependent ◽  
The Individual ◽  
Α Subunits

Voltage-dependent K+ (potassium; Kv) channels are ion channels that critically impact neuronal excitability and function. Four principal α subunits assemble to create a membrane-spanning pore that opens in a voltage-dependent manner to allow the selective passage of K+ ions across the cell membrane. Forty human genes encoding Kv channel α subunits have been identified, and most of them are expressed in the nervous system. The individual Kv subunits display unique cellular and subcellular expression patterns and co-assemble into distinct homo- and hetero-tetrameric channels that differ in their electrophysiological and pharmacological properties, and their sensitivity to dynamic modulation, by cellular signaling pathways. The resulting diversity allows Kv channels to impact all steps in electrical information processing, as well as numerous other aspects of neuronal functions, including those in which they appear to play a non-conducting role. This chapter reviews the current basic knowledge about this large and important family of ion channels.

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.

scholarly journals Effects of exercise training and hypercholesterolemia on adenosine activation of voltage-dependent K+ channels in coronary arterioles

2008 ◽  
Vol 105 (6) ◽  
pp. 1761-1771 ◽  
Author(s):  
Cristine L. Heaps ◽  
Elise C. Jeffery ◽  
Glen A. Laine ◽  
Elmer M. Price ◽  
Douglas K. Bowles
Keyword(s):  
Exercise Training ◽  
Adenylyl Cyclase ◽  
Dependent Manner ◽  
High Cholesterol ◽  
Concentration Dependent Manner ◽  
Kv Channel ◽  
Kv Channels ◽  
Voltage Dependent ◽  
Cyclase Activator ◽  
Coronary Arterioles

Coronary arterioles from hypercholesterolemic swine display attenuated adenosine-mediated vasodilatation that is attributable to the elimination of voltage-dependent K+ (Kv) channel stimulation. For the present study, we tested the hypotheses that exercise training would correct impaired adenosine-induced dilatation in coronary arterioles from hypercholesterolemic pigs through restoration of adenosine activation of Kv channels and that vasodilatation to the receptor-independent adenylyl cyclase activator, forskolin, would also be attenuated in arterioles from hypercholesterolemic pigs. Pigs were randomly assigned to a control (NC) or high-fat, high-cholesterol (HC) diet for 20 wk. Four weeks after the diet was initiated, pigs from both groups were assigned to exercise training (Ex; 5 days/wk for 16 wk) or sedentary (Sed) protocols, resulting in four groups of pigs: NC-Sed, NC-Ex, HC-Sed, and HC-Ex. Arterioles (∼150 μm) from both HC-Sed and HC-Ex pigs displayed impaired adenosine-mediated dilatation that was attributable to the elimination of 4-aminopyridine (4-AP; 1 mM)-sensitive Kv channel activation compared with NC counterparts. Arteriolar smooth muscle whole cell Kv currents were significantly reduced in HC-Sed compared with NC-Sed, although HC-Ex and NC-Ex did not differ. Forskolin-mediated dilatation was attenuated by 4-AP (1 mM) and in a concentration-dependent manner by tetraethylammonium (TEA; 0.1–1 mM) in NC-Sed but not HC-Sed. Further, TEA-sensitive Kv currents were diminished in cells of HC-Sed compared with NC-Sed pigs. Quantitative RT-PCR revealed similar expression levels of Kv3.1 and 3.3 in arterioles of NC-Sed and HC-Sed swine with undetectable expression of Kv1.1, 3.2, and 3.4. Taken together, these results suggest that hypercholesterolemia-mediated attenuation of adenosine-induced vasodilatation in coronary arterioles is not corrected by exercise training and is likely attributable to an impairment in the pathway coupling adenylyl cyclase with a highly TEA-sensitive Kv channel isoform(s).

scholarly journals Molecular Template for a Voltage Sensor in a Novel K+ Channel. III. Functional Reconstitution of a Sensorless Pore Module from a Prokaryotic Kv Channel

2008 ◽  
Vol 132 (6) ◽  
pp. 651-666 ◽  
Author(s):  
Jose S. Santos ◽  
Sergey M. Grigoriev ◽  
Mauricio Montal
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Functional Module ◽  
Ion Selectivity ◽  
Ionic Currents ◽  
K Channel ◽  
Voltage Sensing ◽  
Kv Channel ◽  
Kv Channels ◽  
Conduction Properties

KvLm is a prokaryotic voltage-gated K+ (Kv) channel from Listeria monocytogenes. The sequence of the voltage-sensing module (transmembrane segments S1-S4) of KvLm is atypical in that it contains only three of the eight conserved charged residues known to be deterministic for voltage sensing in eukaryotic Kv's. In contrast, the pore module (PM), including the S4-S5 linker and cytoplasmic tail (linker-S5-P-S6-C-terminus) of KvLm, is highly conserved. Here, the full-length (FL)-KvLm and the KvLm-PM only proteins were expressed, purified, and reconstituted into giant liposomes. The properties of the reconstituted FL-KvLm mirror well the characteristics of the heterologously expressed channel in Escherichia coli spheroplasts: a right-shifted voltage of activation, micromolar tetrabutylammonium-blocking affinity, and a single-channel conductance comparable to that of eukaryotic Kv's. Conversely, ionic currents through the PM recapitulate both the conductance and blocking properties of the FL-KvLm, yet the KvLm-PM exhibits only rudimentary voltage dependence. Given that the KvLm-PM displays many of the conduction properties of FL-KvLm and of other eukaryotic Kv's, including strict ion selectivity, we conclude that self-assembly of the PM subunits in lipid bilayers, in the absence of the voltage-sensing module, generates a conductive oligomer akin to that of the native KvLm, and that the structural independence of voltage sensing and PMs observed in eukaryotic Kv channels was initially implemented by nature in the design of prokaryotic Kv channels. Collectively, the results indicate that this robust functional module will prove valuable as a molecular template for coupling new sensors and to elucidate PM residue–specific contributions to Kv conduction properties.

scholarly journals Voltage-dependent Gating Rearrangements in the Intracellular T1–T1 Interface of a K+ Channel

2006 ◽  
Vol 127 (4) ◽  
pp. 391-400 ◽  
Author(s):  
Guangyu Wang ◽  
Manuel Covarrubias
Keyword(s):  
Electromechanical Coupling ◽  
Channel Gating ◽  
K Channel ◽  
Dependent Manner ◽  
Kv Channels ◽  
Activated State ◽  
Voltage Dependent ◽  
Tightly Coupled ◽  
T1 Domain ◽  
Membrane Spanning

The intracellular tetramerization domain (T1) of most eukaryotic voltage-gated potassium channels (Kv channels) exists as a “hanging gondola” below the transmembrane regions that directly control activation gating via the electromechanical coupling between the S4 voltage sensor and the main S6 gate. However, much less is known about the putative contribution of the T1 domain to Kv channel gating. This possibility is mechanistically intriguing because the T1–S1 linker connects the T1 domain to the voltage-sensing domain. Previously, we demonstrated that thiol-specific reagents inhibit Kv4.1 channels by reacting in a state-dependent manner with native Zn2+ site thiolate groups in the T1–T1 interface; therefore, we concluded that the T1–T1 interface is functionally active and not protected by Zn2+ (Wang, G., M. Shahidullah, C.A. Rocha, C. Strang, P.J. Pfaffinger, and M. Covarrubias. 2005. J. Gen. Physiol. 126:55–69). Here, we co-expressed Kv4.1 channels and auxiliary subunits (KChIP-1 and DPPX-S) to investigate the state and voltage dependence of the accessibility of MTSET to the three interfacial cysteines in the T1 domain. The results showed that the average MTSET modification rate constant (kMTSET) is dramatically enhanced in the activated state relative to the resting and inactivated states (∼260- and ∼47-fold, respectively). Crucially, under three separate conditions that produce distinct activation profiles, kMTSET is steeply voltage dependent in a manner that is precisely correlated with the peak conductance–voltage relations. These observations strongly suggest that Kv4 channel gating is tightly coupled to voltage-dependent accessibility changes of native T1 cysteines in the intersubunit Zn2+ site. Furthermore, cross-linking of cysteine pairs across the T1–T1 interface induced substantial inhibition of the channel, which supports the functionally dynamic role of T1 in channel gating. Therefore, we conclude that the complex voltage-dependent gating rearrangements of eukaryotic Kv channels are not limited to the membrane-spanning core but must include the intracellular T1–T1 interface. Oxidative stress in excitable tissues may perturb this interface to modulate Kv4 channel function.

scholarly journals Kinetic analysis of the action of Leiurus scorpion alpha-toxin on ionic currents in myelinated nerve.

1985 ◽  
Vol 86 (5) ◽  
pp. 739-762 ◽  
Author(s):  
G K Wang ◽  
G Strichartz
Keyword(s):  
Steady State ◽  
Ionic Currents ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Current ◽  
Toxin Concentration ◽  
Toxin Molecule ◽  
Channel Inactivation ◽  
Na Currents ◽  
Voltage Dependent

The effects of a neurotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, on the ionic currents of toad single myelinated fibers were studied under voltage-clamp conditions. Unlike previous investigations using crude scorpion venom, purified Leiurus toxin II alpha at high concentrations (200-400 nM) did not affect the K currents, nor did it reduce the peak Na current in the early stages of treatment. The activation of the Na channel was unaffected by the toxin, the activation time course remained unchanged, and the peak Na current vs. voltage relationship was not altered. In contrast, Na channel inactivation was considerably slowed and became incomplete. As a result, a steady state Na current was maintained during prolonged depolarizations of several seconds. These steady state Na currents had a different voltage dependence from peak Na currents and appeared to result from the opening of previously inactivated Na channels. The opening kinetics of the steady state current were exponential and had rates approximately 100-fold slower than the normal activation processes described for transitions from the resting state to the open state. In addition, the dependence of the peak Na current on the potential of preceding conditioning pulses was also dramatically altered by toxin treatment; this parameter reached a minimal value near a membrane potential of -50 mV and then increased continuously to a "plateau" value at potentials greater than +50 mV. The amplitude of this plateau was dependent on toxin concentration, reaching a maximum value equal to approximately 50% of the peak current; voltage-dependent reversal of the toxin's action limits the amplitude of the plateauing effect. The measured plateau effect was half-maximum at a toxin concentration of 12 nM, a value quite similar to the concentration producing half of the maximum slowing of Na channel inactivation. The results of Hill plots for these actions suggest that one toxin molecule binds to one Na channel. Thus, the binding of a single toxin molecule probably both produces the steady state currents and slows the Na channel inactivation. We propose that Leiurus toxin inhibits the conversion of the open state to inactivated states in a voltage-dependent manner, and thereby permits a fraction of the total Na permeability to remain at membrane potentials where inactivation is normally complete.

Ba2+, TEA+, and quinine effects on apical membrane K+ conductance and maxi K+ channels in gallbladder epithelium

1990 ◽  
Vol 259 (1) ◽  
pp. C56-C68 ◽  
Author(s):  
Y. Segal ◽  
L. Reuss
Keyword(s):  
Apical Membrane ◽  
Membrane Conductance ◽  
Membrane Resistance ◽  
K Channel ◽  
Dependent Manner ◽  
Gallbladder Epithelium ◽  
Concentration Dependent Manner ◽  
K Channels ◽  
Voltage Dependent ◽  
Channel Currents

The apical membrane of Necturus gallbladder epithelium contains a voltage-activated K+ conductance [Ga(V)]. Large-conductance (maxi) K+ channels underlie Ga(V) and account for 17% of the membrane conductance (Ga) under control conditions. We examined the Ba2+, tetraethylammonium (TEA+), and quinine sensitivities of Ga and single maxi K+ channels. Mucosal Ba2+ addition decreased resting Ga in a concentration-dependent manner (65% block at 5 mM) and decreased Ga(V) in a concentration- and voltage-dependent manner. Mucosal TEA+ addition also decreased control Ga (60% reduction at 5 mM). TEA+ block of Ga(V) was more potent and less voltage dependent that Ba2+ block. Maxi K+ channels were blocked by external Ba2+ at millimolar levels and by external TEA+ at submillimolar levels. At 0.3 mM, quinine (mucosal addition) hyperpolarized the cell membranes by 6 mV and reduced the fractional apical membrane resistance by 50%, suggesting activation of an apical membrane K+ conductance. At 1 mM, quinine both activated and blocked K(+)-conductive pathways. Quinine blocked maxi K+ channel currents at submillimolar concentrations. We conclude that 1) Ba2+ and TEA+ block maxi K+ channels and other K+ channels underlying resting Ga; 2) parallels between the Ba2+ and TEA+ sensitivities of Ga(V) and maxi K+ channels support a role for these channels in Ga(V); and 3) quinine has multiple effects on K(+)-conductive pathways in gallbladder epithelium, which are only partially explained by block of apical membrane maxi K+ channels.

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