Heme impairs the ball-and-chain inactivation of potassium channels.

Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.

Properties of Slo1 K+ channels with and without the gating ring.

High-conductance Ca(2+)- and voltage-activated K(+) (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca(2+) and Mg(2+) sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca(2+)- and Mg(2+)-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca(2+) and Mg(2+) activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.

Effect of inserting charged peptide at NH(2)-terminal on N-type inactivation of Kv1.4 channel.

Rapid inactivation of voltage-gated potassium channel plays an important role in shaping the electrical signaling in neurons and other excitable cells. N-type ("ball and chain") inactivation, as the most extensively studied inactivation model, is assumed to be the inactivation mechanism of Kv1.4 channel. The inactivation ball inactivates the channel by interacting with the hydrophobic wall of inner pore and occluding it. Recently, we have proved that the electrostatic interaction between two charged segments in the NH(2)-termainal plays an important role through promoting the inactivation process of the Kv1.4 channel. This study investigates the effect of inserting negatively or positively charged short peptides at NH(2)-terminal on the inactivation of Kv1.4 channel. The results that inserting negatively-charged peptide (either myc or D-peptide) at different sites of NH(2)-terminal, deceleraes inactivation process of Kv1.4 channel to a different extent with inserting site changing and that the mutant Kv1.4-D50 exhibits a more slower inactivation rate than Kv1.4-K50 further identified the role of electrostatic interactions in the "ball and chain" inactivation mechanism.

Electrostatic interaction between inactivation ball and T1-S1 linker region of Kv1.4 channel.

Inactivation of potassium channels plays an important role in shaping the electrical signaling properties of nerve and muscle cells. The rapid inactivation of Kv1.4 has been assumed to be controlled by a "ball and chain" inactivation mechanism. Besides hydrophobic interaction between inactivation ball and the channel's inner pore, the electrostatic interaction has also been proved to participate in the "ball and chain" inactivation process of Kv1.4 channel. Based on the crystal structure of Kv1.2 channel, the acidic T1-S1 linker is indicated to be a candidate interacting with the positively charged hydrophilic region of the inactivation domain. In this study, through mutating the charged residues to amino acids of opposite polar, we identified the electrostatic interaction between the inactivation ball and the T1-S1 linker region of Kv1.4 channel. Inserting negatively charged peptide at the amino terminal of Kv1.4 channel further confirmed the electrostatic interaction between the two regions.

Voltage-dependent gating rearrangements in the intracellular T1-T1 interface of a K+ channel.

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 Zn(2+) site thiolate groups in the T1-T1 interface; therefore, we concluded that the T1-T1 interface is functionally active and not protected by Zn(2+) (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 (k(MTSET)) is dramatically enhanced in the activated state relative to the resting and inactivated states (approximately 260- and approximately 47-fold, respectively). Crucially, under three separate conditions that produce distinct activation profiles, k(MTSET) 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 Zn(2+) 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.

Potassium channel subunit remodeling in rabbits exposed to long-term bradycardia or tachycardia: discrete arrhythmogenic consequences related to differential delayed-rectifier changes.

BACKGROUND: Sustained heart rate abnormalities produce electrical remodeling and susceptibility to arrhythmia. Uncontrolled tachycardia produces heart failure and ventricular tachyarrhythmia susceptibility, whereas bradycardia promotes spontaneous torsade de pointes (TdP). This study compared arrhythmic phenotypes and molecular electrophysiological remodeling produced by tachycardia versus bradycardia in rabbits. METHODS AND RESULTS: We evaluated mRNA and protein expression of subunits underlying rapid (IKr) and slow (IKs) delayed-rectifier and transient-outward K+ currents in ventricular tissues from sinus rhythm control rabbits and rabbits with AV block submitted to 3-week ventricular pacing either at 60 to 90 bpm (bradypaced) or at 350 to 370 bpm (tachypaced). QT intervals at matched ventricular pacing rates were longer in bradypaced than tachypaced rabbits (eg, by approximately 50% at 60 bpm; P<0.01). KvLQT1 and minK mRNA and protein levels were downregulated in both bradypaced and tachypaced rabbits, whereas ERG was significantly downregulated in bradypaced rabbits only. Kv4.3 and Kv1.4 were downregulated by tachypacing only. Patch-clamp experiments showed that IKs was reduced in both but IKr was decreased in bradypaced rabbits only. Continuous monitoring revealed spontaneous TdP in 75% of bradypaced but only isolated ventricular ectopy in tachypaced rabbits. Administration of dofetilide (0.02 mg/kg) to mimic IKr downregulation produced ultimately lethal TdP in all tachypaced rabbits. CONCLUSIONS: Sustained tachycardia and bradycardia downregulate IKs subunits, but bradycardia also suppresses ERG/IKr, causing prominent repolarization delays and spontaneous TdP. Susceptibility of tachycardia/heart failure rabbits to malignant tachyarrhythmias is induced by exposure to IKr blockers. These results point to a crucial role for delayed-rectifier subunit remodeling in TdP susceptibility associated with rate-related cardiac remodeling.

Novel autoantibodies to a voltage-gated potassium channel Kv1.4 in a severe form of myasthenia gravis.

Sera from patients with myasthenia gravis (MG) were screened for autoantibodies to skeletal muscle-specific antigens by immunoprecipitation assay, using rhabdomyosarcoma and leukemia cell lines. Eleven of 61 MG sera immunoprecipitated a rhabdomyosarcoma-specific 70-kDa protein, which was identified as the voltage-gated K+ channel 1.4 (Kv1.4). This antibody specificity was not detected in 30 patients with polymyositis/dermatomyositis, 9 with thymoma alone, or 30 healthy controls. Clinical features associated with anti-Kv1.4 antibody included bulbar involvement, myasthenic crisis, thymoma, myocarditis, and QT prolongation on electrocardiogram. These findings suggest that anti-Kv1.4 antibody is a novel autoantibody associated with a severe MG subset and thymoma.