What keeps Kv channels small? The molecular physiology of modesty.
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ABSTRACT:
SUBMITTER: Sack JT
PROVIDER: S-EPMC4516784 | biostudies-literature | 2015 Aug
REPOSITORIES: biostudies-literature
Publications
What keeps Kv channels small? The molecular physiology of modesty.
Sack Jon T JT Tilley Drew C DC
The Journal of general physiology 20150801 2
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Project description:Background and purposeThe Kv β1.3 subunit modifies the gating and pharmacology of Kv 1.5 channels in a PKC-dependent manner, decreasing channel sensitivity to bupivacaine- and quinidine-mediated blockade. Cardiac Kv 1.5 channels associate with receptor for activated C kinase 1 (RACK1), the Kv β1.3 subunit and different PKC isoforms, resulting in the formation of a functional channelosome. The aim of the present study was to investigate the effects of PKC inhibition on bupivacaine and quinidine block of Kv 1.5 + Kv β1.3 channels.Experimental approachHEK293 cells were transfected with Kv 1.5 + Kv β1.3 channels, and currents were recorded using the whole-cell configuration of the patch-clamp technique. PKC inhibition was achieved by incubating the cells with either calphostin C or bisindolylmaleimide II and the effects of bupivacaine and quinidine were analysed.Key resultsThe voltage-dependent inactivation of Kv 1.5 + Kv β1.3 channels and their pharmacological behaviour after PKC inhibition with calphostin C were similar to those displayed by Kv 1.5 channels alone. Indeed, the IC50 values for bupivacaine were similar in cells whose PKC was inhibited with calphostin C or bisindolylmaleimide II. Similar results were also observed in the presence of quinidine.Conclusions and implicationsThe finding that the voltage-dependence of inactivation and the pharmacology of Kv 1.5 + Kv β1.3 channels after PKC inhibition resembled that observed in Kv 1.5 channels suggests that both processes are dependent on PKC-mediated phosphorylation. These results may have clinical relevance in diseases that are characterized by alterations in kinase activity.
| S-EPMC4294114 | biostudies-literature
Project description:Background and purposeA-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv 4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv 4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv 4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv 4.2/Kv channel-interacting protein 2 (KChIP2) channels.Experimental approachTo screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv 4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv 4.2/KChIP2 channels using a whole-cell patch-clamp technique.Key resultsTyrphostin AG879 selectively and dose-dependently inhibited Kv 4.2 and Kv 4.3 channels. In Kv 4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv 4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons.Conclusion and implicationsAG879 was identified as a selective and potent inhibitor the Kv 4.2 channel. AG879 inhibited Kv 4.2 channels by preferentially interacting with the open state and further accelerating their inactivation.
| S-EPMC4500372 | biostudies-literature
Project description:The molecular mechanisms underlying the activation of Transient Receptor Potential (TRP) ion channels are poorly understood when compared to those of the voltage-activated potassium (Kv) channels. The architectural and pharmacological similarities between the members of these two families of channels suggest that their structure-function relationships may have common features. We explored this hypothesis by replacing previously identified domains and critical structural motifs of the membrane-spanning portions of Kv2.1 with corresponding regions of two TRP channels, TRPM8 and TRPV1. Our results show that the S3b-S4 paddle motif of Kv2.1, but not other domains, can be replaced by the analogous regions of both TRP channels without abolishing voltage-activation. In contrast, replacement of portions of TRP channels with those of Kv2.1 consistently yielded non-functional channels. Taken together, these results suggest that most structural elements within TRP channels and Kv channels are not sufficiently related to allow for the creation of hybrid channels.
| S-EPMC3605605 | biostudies-literature
Project description:The recently determined crystal structure of a chimeric Kv1.2-Kv2.1 Kv channel at 2.4 A resolution motivated this molecular-dynamics simulation study of the chimeric channel and its mutants embedded in a DPPC membrane. For the channel protein, we used two types of C-terminus: E+ and Eo. E+ contains, and Eo lacks, the EGEE residue quartet located distal to the S6 helix. For both E+ and Eo, the following trend was observed: When S4 helices were restrained at the same position as in the x-ray structure (S4high), the S6 gate remained open for 12 ns. The results were similar when the S4 helices were pulled downward 7 A (S4low). However, S4middle (or S4low) facilitated the S6 gate-narrowing for the following mutated channels (shown in order of increasing effect): 1), E395W; 2), E395W-F401A-F402A; and 3), E395W-F401A-F402A-V478W. The amino acid numbering system is that used for the Shaker channel. Even though all four subunits were set at S4low, S6 gate-narrowing was often brought about by movements of only two opposing S6 helices toward the central axis of the pore, resulting in a twofold symmetry-like structure. A free-energy profile analysis over the ion conduction pathway shows that the two opposing S6 helices whose peptide backbones are approximately 10.4 A distant from each other lead to an energetic barrier of approximately 25 kJ/mol. S6 movement was coupled with translocation of the S4-S5 linker toward the central axis of the same subunit, and the coupling was mediated by salt bridges formed between the inner (intracellular side) end of S4 and that of S6. Simulations in which S4 of only one subunit was pulled down to S4low showed that a weak intersubunit coordination is present for S5 movement, whereas the coupling between the S4-S5 linker and S6 is largely an intrasubunit one. In general, whereas subunit-based behavior appears to be dominant and to permit heteromeric conformations of the pore domain, direct intersubunit coupling of S5 or S6 is weak. Therefore, the "concerted transition" of the pore domain that has been predicted based on electrophysiological analyses is likely to be mediated mainly by the dual effects of S4 and the S4-S5 linker; these segments of one subunit can interact with both S5 of the same subunit and that of the adjacent subunit.
| S-EPMC2711363 | biostudies-literature
Project description:Kv channels form voltage-dependent potassium selective pores in the outer cell membrane and are composed out of four α-subunits, each having six membrane-spanning α-helices (S1-S6). The α-subunits tetramerize such that the S5-S6 pore domains co-assemble into a centrally located K(+) pore which is surrounded by four operational voltage-sensing domains (VSD) that are each formed by the S1-S4 segments. Consequently, each subunit is capable of responding to changes in membrane potential and dictates whether the pore should be conductive or not. K(+) permeation through the pore can be sealed off by two separate gates in series: (a) at the inner S6 bundle crossing (BC gate) and (b) at the level of the selectivity filter (SF gate) located at the extracellular entrance of the pore. Within the last years a general consensus emerged that a direct communication between the S4S5-linker and the bottom part of S6 (S6(c)) constitutes the coupling with the VSD thus making the BC gate the main voltage-controllable activation gate. While the BC gate listens to the VSD, the SF changes its conformation depending on the status of the BC gate. Through the eyes of an entering K(+) ion, the operation of the BC gate apparatus can be compared with the iris-like motion of the diaphragm from a camera whereby its diameter widens. Two main gating motions have been proposed to create this BC gate widening: (1) tilting of the helix whereby the S6 converts from a straight α-helix to a tilted one or (2) swiveling of the S6(c) whereby the S6 remains bent. Such motions require a flexible hinge that decouples the pre- and post-hinge segment. Roughly at the middle of the S6 there exists a highly conserved glycine residue and a tandem proline motif that seem to fulfill the role of a gating hinge which allows for tilting/swiveling/rotations of the post-hinge S6 segment. In this review we delineate our current view on the operation of the BC gate for controlling K(+) permeation in Kv channels.
| S-EPMC3440756 | biostudies-literature
Project description:In many voltage-gated K(+) channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na(+) permeability of C-type-inactivated K(+) channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type-inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na(+) tail currents normally observed through C-type-inactivated channels, an effective blockade of the peak Na(+) tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type-inactivated channels. In C-type-deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type-inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na(+) tail of C-type-inactivated channels. Stable binding between the inactivation peptide and the C-type-inactivated state results in slower current decay, and a reduction of the Na(+) tail current magnitude, due to slower transition of channels through the Na(+)-permeable states traversed during recovery from inactivation.
| S-EPMC2234501 | biostudies-literature
Project description:Voltage-activated proton (Hv1) channels are relatives of classical voltage-activated cation channels. In this issue of Neuron, Hong et al. (2013) and Qiu et al. (2013) investigate the functional mechanisms of Hv1 gating and uncover key relationships with Kv channels.
| S-EPMC5111168 | biostudies-literature
Project description:Voltage-gated potassium (Kv) channels control myocardial repolarization. Pore-forming Kvα proteins associate with intracellular Kvβ subunits, which bind pyridine nucleotides with high affinity and differentially regulate channel trafficking, plasmalemmal localization and gating properties. Nevertheless, it is unclear how Kvβ subunits regulate myocardial K+ currents and repolarization. Here, we tested the hypothesis that Kvβ2 subunits regulate the expression of myocardial Kv channels and confer redox sensitivity to Kv current and cardiac repolarization. Co-immunoprecipitation and in situ proximity ligation showed that in cardiac myocytes, Kvβ2 interacts with Kv1.4, Kv1.5, Kv4.2, and Kv4.3. Cardiac myocytes from mice lacking Kcnab2 (Kvβ2-/-) had smaller cross sectional areas, reduced sarcolemmal abundance of Kvα binding partners, reduced Ito, IK,slow1, and IK,slow2 densities, and prolonged action potential duration compared with myocytes from wild type mice. These differences in Kvβ2-/- mice were associated with greater P wave duration and QT interval in electrocardiograms, and lower ejection fraction, fractional shortening, and left ventricular mass in echocardiographic and morphological assessments. Direct intracellular dialysis with a high NAD(P)H:NAD(P)+ accelerated Kv inactivation in wild type, but not Kvβ2-/- myocytes. Furthermore, elevated extracellular levels of lactate increased [NADH]i and prolonged action potential duration in wild type cardiac myocytes and perfused wild type, but not Kvβ2-/-, hearts. Taken together, these results suggest that Kvβ2 regulates myocardial electrical activity by supporting the functional expression of proteins that generate Ito and IK,slow, and imparting redox and metabolic sensitivity to Kv channels, thereby coupling cardiac repolarization to myocyte metabolism.
| S-EPMC7344177 | biostudies-literature
Project description:Background and purposeKCNQ-encoded voltage-dependent potassium channels (Kv 7) are involved in the regulation of vascular tone. In this study we evaluated the influence of Kv 7 channel activation on smooth muscle relaxation in rat penile arteries and corpus cavernosum from normal and spontaneously hypertensive, heart failure-prone (SHHF) rats - a rat model of human metabolic syndrome.Experimental approachQuantitative PCR and immunohistochemistry were used to determine the expression of KCNQ isoforms in penile tissue. Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from normal and SHHF rats.Key resultsTranscripts for KCNQ3, KCNQ4 and KCNQ5 were detected in penile arteries and corpus cavernosum. KCNQ1 was only found in corpus cavernosum. Immunofluorescence signals to Kv 7.4 and Kv 7.5 were found in penile arteries, penile veins and corpus cavernosum. The Kv 7.2-7.5 activators, ML213 and BMS204352, relaxed pre-contracted penile arteries and corpus cavernosum independently of nitric oxide synthase or endothelium-derived hyperpolarization. Relaxations to sildenafil, a PDE5 inhibitor, and sodium nitroprusside (SNP), an nitric oxide donor, were reduced by blocking Kv 7 channels with linopirdine in penile arteries and corpus cavernosum. In SHHF rat penile arteries and corpus cavernosum, relaxations to ML213 and BMS204352 were attenuated, and the blocking effect of linopirdine on sildenafil-induced and SNP-induced relaxations reduced. KCNQ3, KCNQ4 and KCNQ5 were down-regulated, and KCNQ1 was up-regulated in corpus cavernosum from SHHF rats. KCNQ1-5 transcripts remained unchanged in penile arteries from SHHF rats.Conclusions and implicationsThese data suggest that Kv 7 channels play a role in erectile function and contribute to the pathophysiology of erectile dysfunction, an early indicator of cardiovascular disease.
| S-EPMC4831315 | biostudies-literature
Project description:BackgroundPolycystin-1 (PC1) is a transmembrane protein originally identified in autosomal dominant polycystic kidney disease where it regulates the calcium-permeant cation channel polycystin-2. Autosomal dominant polycystic kidney disease patients develop renal failure, hypertension, left ventricular hypertrophy, and diastolic dysfunction, among other cardiovascular disorders. These individuals harbor PC1 loss-of-function mutations in their cardiomyocytes, but the functional consequences are unknown. PC1 is ubiquitously expressed, and its experimental ablation in cardiomyocyte-specific knockout mice reduces contractile function. Here, we set out to determine the pathophysiological role of PC1 in cardiomyocytes.MethodsWild-type and cardiomyocyte-specific PC1 knockout mice were analyzed by echocardiography. Excitation-contraction coupling was assessed in isolated cardiomyocytes and human embryonic stem cell-derived cardiomyocytes, and functional consequences were explored in heterologous expression systems. Protein-protein interactions were analyzed biochemically and by means of ab initio calculations.ResultsPC1 ablation reduced action potential duration in cardiomyocytes, decreased Ca2+ transients, and myocyte contractility. PC1-deficient cardiomyocytes manifested a reduction in sarcoendoplasmic reticulum Ca2+ stores attributable to a reduced action potential duration and sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) activity. An increase in outward K+ currents decreased action potential duration in cardiomyocytes lacking PC1. Overexpression of full-length PC1 in HEK293 cells significantly reduced the current density of heterologously expressed Kv4.3, Kv1.5 and Kv2.1 potassium channels. PC1 C terminus inhibited Kv4.3 currents to the same degree as full-length PC1. Additionally, PC1 coimmunoprecipitated with Kv4.3, and a modeled PC1 C-terminal structure suggested the existence of 2 docking sites for PC1 within the N terminus of Kv4.3, supporting a physical interaction. Finally, a naturally occurring human mutant PC1R4228X manifested no suppressive effects on Kv4.3 channel activity.ConclusionsOur findings uncover a role for PC1 in regulating multiple Kv channels, governing membrane repolarization and alterations in SERCA activity that reduce cardiomyocyte contractility.
| S-EPMC6733647 | biostudies-literature