14-3-3 amplifies and prolongs adrenergic stimulation of HERG K+ channel activity

Acute stress provokes lethal cardiac arrhythmias in the hereditary long QT syndrome. Here we provide a novel molecular mechanism linking beta-adrenergic signaling and altered human ether-a-go-go related gene (HERG) channel activity. Stress stimulates beta-adrenergic receptors, leading to cAMP elevations that can regulate HERG K+ channels both directly and via phosphorylation by cAMP-dependent protein kinase (PKA). We show that HERG associates with 14-3-3epsilon to potentiate cAMP/PKA effects upon HERG. The binding of 14-3-3 occurs simultaneously at the N- and C-termini of the HERG channel. 14-3-3 accelerates and enhances HERG activation, an effect that requires PKA phosphorylation of HERG and dimerization of 14-3-3. The interaction also stabilizes the lifetime of the PKA-phosphorylated state of the channel by shielding the phosphates from cellular phosphatases. The net result is a prolongation of the effect of adrenergic stimulation upon HERG activity. Thus, 14-3-3 interactions with HERG may provide a unique mechanism for plasticity in the control of membrane excitability and cardiac rhythm.     

Molecular and functional characterization of common polymorphisms in HERG (KCNH2) potassium channels

Long QT syndrome (LQTS) is a cardiac repolarization disorder that can lead to arrhythmias and sudden death. Chromosome 7-linked inherited LQTS (LQT2) is caused by mutations in human ether-a-go-go-related gene (HERG; KCNH2), whereas drug-induced LQTS is caused primarily by HERG channel block. Many common polymorphisms are functionally silent and have been traditionally regarded as benign and without physiological consequence. However, the identification of common nonsynonymous single nucleotide polymorphisms (nSNPs; i.e., amino-acid coding variants) with functional phenotypes in the SCN5A Na(+) channel and MiRP1 K(+) channel beta-subunit have challenged this viewpoint. In this report, we test the hypothesis that common missense HERG polymorphisms alter channel physiology. Comprehensive mutational analysis of HERG was performed on genomic DNA derived from a population-based cohort of sudden infant death syndrome and two reference allele cohorts derived from 100 African American and 100 Caucasian individuals. Amino acid-encoding variants were considered common polymorphisms if they were present in at least two of the three study cohorts with an allelic frequency >0.5%. Four nSNPs were identified: K897T, P967L, R1047L, and Q1068R. Wild-type (WT) and polymorphic channels were heterologously expressed in human embryonic kidney cells, and biochemical and voltage-clamp techniques were used to characterize their functional properties. All channel types were processed similarly, but several electrophysiological differences were identified: 1) K897T current density was lower than the other polymorphic channels; 2) K897T channels activated at more negative potentials than WT and R1047L; 3) K897T and Q1068R channels inactivated and recovered from inactivation faster than WT, P967L, and R1047L channels; and 4) K897T channels showed subtle differences compared with WT channels when stimulated with an action potential waveform. In contrast to K897T and Q1068R channels, P967L and R1047L channels were electrophysiologically indistinguishable from WT channels. All HERG channels had similar sensitivity to block by cisapride. Therefore, some HERG polymorphic channels are electrophysiologically different from WT channels.     

A-Kinase Anchoring Protein Targeting of Protein Kinase A and Regulation of HERG Channels

Adrenergic stimulation of the heart initiates a signaling cascade in cardiac myocytes that increases the concentration of cAMP. Although cAMP elevation may occur over a large area of a target-organ cell, its effects are often more restricted due to local concentration of its main effector, protein kinase A (PKA), through A-kinase anchoring proteins (AKAPs). The HERG potassium channel, which produces the cardiac rapidly activating delayed rectifying K(+) current (I (Kr)), is a target for cAMP/PKA regulation. PKA regulation of the current may play a role in the pathogenesis of hereditary and acquired abnormalities of the channel leading to cardiac arrhythmia. We examined the possible role for AKAP-mediated regulation of HERG channels. Here, we report that the PKA-RII-specific AKAP inhibitory peptide AKAP-IS perturbs the distribution of PKA-RII and diminishes the PKA-dependent phosphorylation of HERG protein. The functional consequence of AKAP-IS is a reversal of cAMP-dependent regulation of HERG channel activity. In further support of AKAP-mediated targeting of kinase to HERG, PKA activity was coprecipitated from HERG expressed in HEK cells. Velocity gradient centrifugation of solubilized porcine cardiac membrane proteins showed that several PKA-RI and PKA-RII binding proteins cosediment with ERG channels. A physical association of HERG with several specific AKAPs with known cardiac expression, however, was not demonstrable in heterologous cotransfection studies. These results suggest that one or more AKAP(s) targets PKA to HERG channels and may contribute to the acute regulation of I (Kr) by cAMP.     

Identification of a COOH-terminal segment involved in maturation and stability of human ether-a-go-go-related gene potassium channels

Mutations in the potassium channel encoded by the human ether-a-go-go-related gene (HERG) have been linked to the congenital long QT syndrome (LQTS), a cardiac disease associated with an increased preponderance of ventricular arrhythmias and sudden death. The COOH terminus of HERG harbors a large number of LQTS mutations and its removal prevents functional expression for reasons that remain unknown. In this study, we show that the COOH terminus of HERG is required for normal trafficking of the ion channel. We have identified a region critical for trafficking between residues 860 and 899 that includes a novel missense mutation at amino acid 861 (HERGN861I). Truncations or deletion of residues 860-899, characterized in six different expression systems including a cardiac cell line, resulted in decreased expression levels and an absence of the mature glycosylated form of the HERG protein. Deletion of this region did not interfere with the formation of tetramers but caused retention of the assembled ion channels within the endoplasmic reticulum. Consequently, removal of residues 860-899 resulted in the absence of the ion channels from the cell surface and a more rapid turnover rate than the wild type channels, which was evident very early in biogenesis. This study reveals a novel role of the COOH terminus in the normal biogenesis of HERG channels and suggests defective trafficking as a common mechanism for abnormal channel function resulting from mutations of critical COOH-terminal residues, including the LQTS mutant HERGN861I.     

Structure of the HERG K+ channel S5P extracellular linker: role of an amphipathic alpha-helix in C-type inactivation

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585-Ile593 and Gly604-Tyr611 of the channel. The Trp585-Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604-Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.     

RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte expression studies

The physiological properties of most ion channels are defined experimentally by functional expression of their pore-forming alpha subunits in Xenopus laevis oocytes. Here, we cloned a family of Xenopus KCNE genes that encode MinK-related peptide K(+) channel beta subunits (xMiRPs) and demonstrated their constitutive expression in oocytes. Electrophysiological analysis of xMiRP2 revealed that when overexpressed this gene modulates human cardiac K(+) channel alpha subunits HERG (human ether-a-go-go-related gene) and KCNQ1 by suppressing HERG currents and removing the voltage dependence of KCNQ1 activation. The ability of endogenous levels of xMiRP2 to contribute to the biophysical attributes of overexpressed mammalian K(+) channels in oocyte studies was assessed next. Injection of an xMiRP2 sequence-specific short interfering RNA (siRNA) oligo reduced endogenous xMiRP2 expression 5-fold, whereas a control siRNA oligo had no effect, indicating the effectiveness of the RNA interference technique in Xenopus oocytes. The functional effects of endogenous xMiRP2 silencing were tested using electrophysiological analysis of heterologously expressed HERG channels. The RNA interference-mediated reduction of endogenous xMiRP2 expression increased macroscopic HERG current as much as 10-fold depending on HERG cRNA concentration. The functional effects of human MiRP1 (hMiRP1)/HERG interaction were also affected by endogenous xMiRP2. At high HERG channel density, at which the effects of endogenous xMiRP2 are minimal, hMiRP1 reduced HERG current. At low HERG current density, hMiRP1 paradoxically up-regulated HERG current, a result consistent with hMiRP1 rescuing HERG from suppression by endogenous xMiRP2. Thus, endogenous Xenopus MiRP subunits contribute to the base-line properties of K(+) channels like HERG in oocyte expression studies, which could explain expression level- and expression system-dependent variation in K(+) channel function.     

The HERG K<Superscript>+</Superscript> channel: progress in understanding the molecular basis of its unusual gating kinetics

The HERG K+ channel has very unusual kinetic behaviour that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarisation as well as in preventing lethal ventricular arrhythmias. Extensive mutagenesis of the HERG K+ channel has allowed identification of which regions of the channel are important for the unusual kinetic behaviour of the channel. Furthermore, structural studies on scorpion toxins that potently inhibit HERG are beginning to provide clues as to the structural differences between HERG and other voltage-gated K+ channels.     

Characterisation of recombinant HERG K+ channel blockade by the Class Ia antiarrhythmic drug procainamide

Class Ia antiarrhythmic drugs, including procainamide (PROC), are associated with cardiac sodium channel blockade, delayed ventricular repolarisation and with a risk of ventricular pro-arrhythmia. The HERG K(+) channel is frequently linked to drug-induced pro-arrhythmia. Therefore, in this study, interactions between PROC and HERG K(+) channels were investigated, with particular reference to potency and mechanism of drug action. Whole-cell patch-clamp recordings of HERG current (I(HERG)) were made at 37 degrees C from human embryonic kidney (HEK 293) cells stably expressing the HERG channel. Following activating pulses to +20 mV, I(HERG) tails were inhibited by PROC with an IC(50) value of approximately 139 microM. I(HERG) blockade was found to be both time- and voltage-dependent, demonstrating contingency upon HERG channel gating. However, I(HERG) inhibition by PROC was relieved by depolarisation to a highly positive membrane potential (+80 mV) that favoured HERG channel inactivation. These data suggest that PROC inhibits the HERG K(+) channel by a primarily 'open' or 'activated' channel state blocking mechanism and that avidity of drug-binding is decreased by extensive I(HERG) inactivation. The potency of I(HERG) blockade by PROC is much lower than for other Class Ia agents that have been studied previously under analogous conditions (quinidine and disopyramide), although the blocking mechanism appears similar. Thus, differences between the chemical structure of PROC and other Class Ia antiarrhythmic drugs may help provide insight into chemical determinants of blocking potency for agents that bind to open/activated HERG channels.     

Vasoactive intestinal peptide activates Ca2(+)-dependent K+ channels through a cAMP pathway in mouse lacrimal cells

The action of vasoactive intestinal peptide (VIP) on Ca2(+)-dependent K+ currents, in dissociated mouse lacrimal cells, was investigated using patch clamp techniques. In whole cell recordings, VIP (10-100 pM) increased the magnitude of the Ca2(+)-dependent K+ current. In single channel recordings, VIP increased the fraction of time the large charybdotoxin-sensitive Ca2(+)-activated K+ channel spent in the open state. The activity of this channel was also increased by adding forskolin or 8-bromo cAMP to the bath. Additionally, application of either cAMP or catalytic subunit of cAMP-dependent protein kinase directly to the cytoplasmic surface of excised inside out patches reversibly lengthened the time Ca2(+)-activated K+ channels spent in the open state. These data suggest that VIP stimulates Ca2(+)-activated K+ channels by a cAMP-dependent pathway in mouse lacrimal acinar cells.     

Molecular determinants of voltage-dependent human ether-a-go-go related gene (HERG) K+ channel block

The structural determinants for the voltage-dependent block of ion channels are poorly understood. Here we investigate the voltage-dependent block of wild-type and mutant human ether-a-go-go related gene (HERG) K(+) channels by the antimalarial compound chloroquine. The block of wild-type HERG channels expressed in Xenopus oocytes was enhanced as the membrane potential was progressively depolarized. The IC(50) was 8.4 +/- 0.9 microm when assessed during 4-s voltage clamp pulses to 0 mV. Chloroquine also slowed the apparent rate of HERG deactivation, reflecting the inability of drug-bound channels to close. Mutation to alanine of aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of HERG greatly reduced the potency of channel block by chloroquine (IC(50) > 1 mm at 0 mV). However, mutation of Tyr-652 also altered the voltage dependence of the block. In contrast to wild-type HERG, block of Y652A HERG channels was diminished by progressive membrane depolarization, and complete relief from block was observed at +40 mV. HERG channel block was voltage-independent when the hydroxyl group of Tyr-652 was removed by mutating the residue to Phe. Together these findings indicate a critical role for Tyr-652 in voltage-dependent block of HERG channels. Molecular modeling was used to define energy-minimized dockings of chloroquine to the central cavity of HERG. Our experimental findings and modeling suggest that chloroquine preferentially blocks open HERG channels by cation-pi and pi-stacking interactions with Tyr-652 and Phe-656 of multiple subunits.     

Deletion of protein kinase A phosphorylation sites in the HERG potassium channel inhibits activation shift by protein kinase A.

We investigated the role of protein kinase A (PKA) in regulation of the human ether-a-go-go-related gene (HERG) potassium channel activation. HERG clones with single mutations destroying one of four consensus PKA phosphorylation sites (S283A, S890A, T895A, S1137A), as well as one clone carrying all mutations with no PKA phosphorylation sites (HERG 4M) were constructed. These clones were expressed heterologously in Xenopus oocytes, and HERG potassium currents were measured with the two microelectrode voltage clamp technique. Application of the cAMP-specific phosphodiesterase (PDE IV) inhibitor Ro-20-1724 (100 microM), which results in an increased cAMP level and PKA stimulation, induced a reduction of HERG wild type outward currents by 19.1% due to a shift in the activation curve of 12.4 mV. When 100 microM Ro-20-1724 was applied to the HERG 4M channel, missing all PKA sites, there was no significant shift in the activation curve, and the current amplitude was not reduced. Furthermore, the adenylate cyclase activator forskolin that leads to PKA activation (400 microM, 60 min), shifted HERG wild type channel activation by 14.1 mV and reduced currents by 39.9%, whereas HERG 4M channels showed only a small shift of 4.3 mV and a weaker current reduction of 22.3%. We conclude that PKA regulates HERG channel activation, and direct phosphorylation of the HERG channel protein has a functional role that may be important in regulation of cardiac repolarization.     

Analysis of the cyclic nucleotide binding domain of the HERG potassium channel and interactions with KCNE2

Mutations in the cyclic nucleotide binding domain (CNBD) of the human ether-a-go-go-related gene (HERG) K+ channel are associated with LQT2, a form of hereditary Long QT syndrome (LQTS). Elevation of cAMP can modulate HERG K+ channels both by direct binding and indirect regulation through protein kinase A. To assess the physiological significance of cAMP binding to HERG, we introduced mutations to disrupt the cyclic nucleotide binding domain. Eight mutants including two naturally occurring LQT2 mutants V822M and R823W were constructed. Relative cAMP binding capacity was reduced or absent in CNBD mutants. Mutant homotetramers carry little or no K+ current despite normal protein abundance and surface expression. Co-expression of mutant and wild-type HERG resulted in currents with altered voltage dependence but without dominant current suppression. The data from co-expression of V822M and wild-type HERG best fit a model where one normal subunit within a tetramer allows nearly normal current expression. The presence of KCNE2, an accessory protein that associates with HERG, however, conferred a partially dominant current suppression by CNBD mutants. Thus KCNE2 plays a pivotal role in determining the phenotypic severity of some forms of LQT2, which suggests that the CNBD of HERG may be involved in its interaction with KCNE2.     

A model for identifying HERG K+ channel blockers

Acquired long QT syndrome (LQTS) occurs frequently as a side effect of blockade of cardiac HERG K(+) channels by commonly used medications. A large number of structurally diverse compounds have been shown to inhibit K(+) current through HERG. There is considerable interest in developing in silico tools to filter out potential HERG blockers early in the drug discovery process. We describe a binary classification model that combines a 2D topological similarity filter with a 3D pharmacophore ensemble procedure to discriminate between HERG actives and inactives with an overall accuracy of 82%, with false negative and false positive rates of 29% and 15%, respectively. This model should be generally applicable in virtual library counterscreening against HERG.     

The binding site for channel blockers that rescue misprocessed human long QT syndrome type 2 ether-a-gogo-related gene (HERG) mutations.

Mutations in the human ether-a-gogo-related gene (HERG) K(+) channel gene cause chromosome 7-linked long QT syndrome type 2 (LQT2), which is characterized by a prolonged QT interval in the electrocardiogram and an increased susceptibility to life-threatening cardiac arrhythmias. LQT2 mutations produce loss-of-function phenotypes and reduce I(Kr) currents either by the heteromeric assembly of non- or malfunctioning channel subunits with wild type subunits at the cell surface or by retention of misprocessed mutant HERG channels in the endoplasmic reticulum. Misprocessed mutations often encode for channel proteins that are functional upon incorporation into the plasma membrane. As a result the pharmacological correction of folding defects and restoration of protein function are of considerable interest. Here we report that the trafficking-deficient pore mutation HERG G601S was rescued by a series of HERG channel blockers that increased cell surface expression. Rescue by these pharmacological chaperones varied directly with their blocking potency. We used structure-activity relationships and site-directed mutagenesis to define the binding site of the pharmacological chaperones. We found that binding occurred in the inner cavity and correlated with hydrophobicity and cationic charge. Rescue was domain-restricted because the trafficking of two misprocessed mutations in the C terminus, HERG F805C and HERG R823W, was not restored by channel blockers. Our findings represent a first step toward the design of pharmacological chaperones that will rescue HERG K(+) channels without block.     

Progesterone impairs human ether-a-go-go-related gene (HERG) trafficking by disruption of intracellular cholesterol homeostasis

The prolongation of QT intervals in both mothers and fetuses during the later period of pregnancy implies that higher levels of progesterone may regulate the function of the human ether-a-go-go-related gene (HERG) potassium channel, a key ion channel responsible for controlling the length of QT intervals. Here, we studied the effect of progesterone on the expression, trafficking, and function of HERG channels and the underlying mechanism. Treatment with progesterone for 24 h decreased the abundance of the fully glycosylated form of the HERG channel in rat neonatal cardiac myocytes and HERG-HEK293 cells, a cell line stably expressing HERG channels. Progesterone also concentration-dependently decreased HERG current density, but had no effect on voltage-gated L-type Ca(2+) and K(+) channels. Immunofluorescence microscopy and Western blot analysis show that progesterone preferentially decreased HERG channel protein abundance in the plasma membrane, induced protein accumulation in the dilated endoplasmic reticulum (ER), and increased the protein expression of C/EBP homologous protein, a hallmark of ER stress. Application of 2-hydroxypropyl-β-cyclodextrin (a sterol-binding agent) or overexpression of Rab9 rescued the progesterone-induced HERG trafficking defect and ER stress. Disruption of intracellular cholesterol homeostasis with simvastatin, imipramine, or exogenous application of cholesterol mimicked the effect of progesterone on HERG channel trafficking. Progesterone may impair HERG channel folding in the ER and/or block its trafficking to the Golgi complex by disrupting intracellular cholesterol homeostasis. Our findings may reveal a novel molecular mechanism to explain the QT prolongation and high risk of developing arrhythmias during late pregnancy.     

High affinity HERG K(+) channel blockade by the antiarrhythmic agent dronedarone: resistance to mutations of the S6 residues Y652 and F656

Pharmacological inhibition of human-ether-a-go-go-related gene (HERG) K(+) channels by structurally and therapeutically diverse drugs is associated with the 'acquired' form of long QT syndrome and with potentially lethal cardiac arrhythmias. Two aromatic amino-acid residues (Y652 and F656) on the inner (S6) helices are considered to be key constituents of a high affinity drug binding site within the HERG channel pore cavity. Using wild-type (WT) and mutant HERG channels expressed in mammalian cell lines, we have investigated HERG channel current (I(HERG)) blockade at 37+/-1 degrees C by dronedarone (DRONED), a non-iodinated analogue of the Class III antiarrhythmic agent amiodarone (AMIOD). Under our conditions WT I(HERG) tails, measured at -40 mV following activating pulses to +30 mV, were blocked with IC(50) values of approximately 59 and 70 nM for DRONED and AMIOD, respectively. I(HERG) inhibition by DRONED was contingent upon channel gating, with block developing rapidly on membrane depolarization, but with no preference for activated over inactivated channels. High external [K(+)] (94 mM) reduced the potency of I(HERG) inhibition by both DRONED and AMIOD. Strikingly, mutagenesis to alanine of the S6 residue F656 (F656A) failed to eliminate blockade by both DRONED and AMIOD, whilst Y652A had comparatively little effect on DRONED but some effect on AMIOD. These findings demonstrate that high affinity drug blockade of I(HERG) can occur without a strong dependence on the Y652 and F656 aromatic amino-acid residues.     

Trapping of a methanesulfonanilide by closure of the HERG potassium channel activation gate

Deactivation of voltage-gated potassium (K(+)) channels can slow or prevent the recovery from block by charged organic compounds, a phenomenon attributed to trapping of the compound within the inner vestibule by closure of the activation gate. Unbinding and exit from the channel vestibule of a positively charged organic compound should be favored by membrane hyperpolarization if not impeded by the closed gate. MK-499, a methanesulfonanilide compound, is a potent blocker (IC(50) = 32 nM) of HERG K(+) channels. This bulky compound (7 x 20 A) is positively charged at physiological pH. Recovery from block of HERG channels by MK-499 and other methanesulfonanilides is extremely slow (Carmeliet 1992; Ficker et al. 1998), suggesting a trapping mechanism. We used a mutant HERG (D540K) channel expressed in Xenopus oocytes to test the trapping hypothesis. D540K HERG has the unusual property of opening in response to hyperpolarization, in addition to relatively normal gating and channel opening in response to depolarization (Sanguinetti and Xu 1999). The hyperpolarization-activated state of HERG was characterized by long bursts of single channel reopening. Channel reopening allowed recovery from block by 2 microM MK-499 to occur with time constants of 10.5 and 52.7 s at -160 mV. In contrast, wild-type HERG channels opened only briefly after membrane hyperpolarization, and thus did not permit recovery from block by MK-499. These findings provide direct evidence that the mechanism of slow recovery from HERG channel block by methanesulfonanilides is due to trapping of the compound in the inner vestibule by closure of the activation gate. The ability of HERG channels to trap MK-499, despite its large size, suggests that the vestibule of this channel is larger than the well studied Shaker K(+) channel.     

Cloning and functional characterization of the smooth muscle ether-a-go-go-related gene K+ channel. Potential role of a conserved amino acid substitution in the S4 region

The human ether-a-go-go-related gene (HERG) product forms the pore-forming subunit of the delayed rectifier K(+) channel in the heart. Unlike the cardiac isoform, the erg K(+) channels in native smooth muscle demonstrate gating properties consistent with a role in maintaining resting potential. We have cloned the smooth muscle isoform of HERG, denoted as erg1-sm, from human and rabbit colon. erg1-sm is truncated by 101 amino acids in the C terminus due to a single nucleotide deletion in the 14th exon. Sequence alignment against HERG showed a substitution of alanine for valine in the S4 domain. When expressed in Xenopus oocytes, erg1-sm currents had much faster activation and deactivation kinetics compared with HERG. Step depolarization positive to -20 mV consistently produced a transient outward component. The threshold for activation of erg1-sm was -60 mV and steady-state conductance was approximately 10-fold greater than HERG near the resting potential of smooth muscle. Site-directed mutagenesis of alanine to valine in the S4 region of erg1-sm converted many of the properties to that of the cardiac HERG, including shifts in the voltage dependence of activation and slowing of deactivation. These studies define the functional role of a novel isoform of the ether-a-go-go-related gene K(+) channel in smooth muscle.     

Interactions between S4-S5 linker and S6 transmembrane domain modulate gating of HERG K+ channels

Outward movement of the voltage sensor is coupled to activation in voltage-gated ion channels; however, the precise mechanism and structural basis of this gating event are poorly understood. Potential insight into the coupling mechanism was provided by our previous finding that mutation to Lys of a single residue (Asp(540)) located in the S4-S5 linker endowed HERG (human ether-a-go-go-related gene) K(+) channels with the unusual ability to open in response to membrane depolarization and hyperpolarization in a voltage-dependent manner. We hypothesized that the unusual hyperpolarization-induced gating occurred through an interaction between Lys(540) and the C-terminal end of the S6 domain, the region proposed to form the activation gate. Therefore, we mutated six residues located in this region of S6 (Ile(662)-Tyr(667)) to Ala in D540K HERG channels. Mutation of Arg(665), but not the other five residues, prevented hyperpolarization-dependent reopening of D540K HERG channels. Mutation of Arg(665) to Gln or Asp also prevented reopening. In addition, D540R and D540K/R665K HERG reopened in response to hyperpolarization. Together these findings suggest that a single residue (Arg(665)) in the S6 domain interacts with Lys(540) by electrostatic repulsion to couple voltage sensing to hyperpolarization-dependent opening of D540K HERG K(+) channels. Moreover, our findings suggest that the C-terminal ends of S4 and S6 are in close proximity at hyperpolarized membrane potentials.     

A novel calcium-sensing domain in the BK channel.

The high-conductance Ca2+-activated K+ channel (mSlo) plays a vital role in regulating calcium entry in many cell types. mSlo channels behave like voltage-dependent channels, but their voltage range of activity is set by intracellular free calcium. The mSlo subunit has two parts: a "core" resembling a subunit from a voltage-dependent K+ channel, and an appended "tail" that plays a role in calcium sensing. Here we present evidence for a site on the tail that interacts with calcium. This site, the "calcium bowl," is a novel calcium-binding motif that includes a string of conserved aspartate residues. Mutations of the calcium bowl fall into two categories: 1) those that shift the position of the G-V relation a similar amount at all [Ca2+], and 2) those that shift the position of the G-V relation only at low [Ca2+]. None of these mutants alters the slope of the G-V curve. These mutant phenotypes are apparent in calcium ion, but not in cadmium ion, where mutant and wild type are indistinguishable. This suggests that the calcium bowl is sensitive to calcium ion, but insensitive to cadmium ion. The presence and independence of a second calcium-binding site is inferred because channels still respond to increasing levels of [Ca2+] or [Cd2+], even when the calcium bowl is mutationally deleted. Thus a low level of activation in the absence of divalent cations is identical in mutant and wild-type channels, possibly because of activation of this second Ca2+-binding site.