Saman Rezazadeh

Rb+ flux through hERG channels affects the potency of channel blocking drugs: Correlation with data obtained using a high-throughput Rb+ efflux assay

The nonradioactive Rb+ efflux assay has become a reliable and efficient high-throughput hERG screening method, but it is limited by its low sensitivity for potent hERG blockers. Using the patch clamp technique, the authors found that the low sensitivity is due in part to the use of Rb+ as the permeating cation in the assay. The affinities of the drugs measured by patch clamp technique in the presence of Rb+ were 3- to 10-fold lower than when measured by the same method in the presence of K+ ions. The apparent affinity of the drugs decreased even further when monitored bytheRb+ efflux assay. It was also observed that Rb+ had minimal effects on the activation properties of channels while there was a significant change in the half-inactivation potential. This voltage shift reduces hERG channel inactivation at efflux assay potentials, and will reduce the affinity of hERG-blocking drugs that bind to inactivated states of the channel. In combination with the effects of elevated extracellular ion concentrations, it is likely that Rb+ modulation of hERG channel inactivation is largely responsible for the reduced drug potencies observed in the Rb+ efflux assay.

Flux assays in high throughput screening of ion channels in drug discovery.

Ion channels have been identified as therapeutic targets in various disorders, such as cardiovascular disease, neurological disease, and cystic fibrosis. Flux assays to detect functional ionic flux through ion channels are becoming increasingly popular as tools for screening compounds. In an optimized flux assay, modulation of ion channel activity may produce readily detectable changes in radiolabeled or nonradiolabeled ionic flux. Technologies based on flux assays are currently available in a fully automated high throughput format for efficient screening. This application offers sensitive, precise, and reproducible measurements giving accurate drug rank orders matching those of patch clamp data. Conveniently, the flux assay is amenable to adaptation for different ion channels, such as potassium, sodium, calcium, and chloride channels, by using suitable tracer ions. The nonradiolabeled rubidium-based flux assay coupled with the ion channel reader (ICR) technology has become very successful in ion channel activity analysis and is emerging as a popular technique in modern drug discovery.

A direct demonstration of closed-state inactivation of K+ channels at low pH

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K+ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.

A KCNQ1 V205M missense mutation causes a high rate of long QT syndrome in a First Nations community of northern British Columbia: a community-based approach to understanding the impact.

Purpose: Hereditary long QT syndrome is named for a prolonged QT interval reflecting predisposition to ventricular arrhythmias and sudden death. A high rate in a remote, northern Canadian First Nations community was brought to attention.Methods: Two severely affected index cases and 122 relatives were ascertained using community-based participatory research principles. Genetic sequencing of five known genes responsible for long QT syndrome was carried out on the index cases, leading to the identification of a novel missense mutation. Functional properties of the identified mutation were studied in transfected mouse ltk- cells using whole cell patch clamp techniques. Corrected QT interval measurements were obtained from participants and subsequent genotyping of relatives was carried out.Results: In the two index cases, a novel missense mutation (V205M) was identified in the S3 transmembrane helix of KvLQT1, the pore forming domain of the IKs channel complex. In transfected mouse ltk-cells the V205M mutation suppressed IKs by causing a dramatic depolarizing shift in activation voltage coupled with acceleration of channel deactivation. Twenty-two mutation carriers had a significantly higher mean corrected QT interval than noncarriers (465 ± 28 milliseconds vs. 434 ± 26 milliseconds, P < 0.0001); however, 30% of carriers had a corrected QT interval below 440 milliseconds.Conclusion: A novel KCNQ1 mutation in this founder population likely confers increased susceptibility to arrhythmias because of decreased IKs current. Even with a common mutation within a relatively homogenous population, clinical expression remains variable, exemplifying the multifactorial nature of long QT syndrome, and supporting the difficulty of definitive diagnosis without genetic testing. A community participatory approach enabled a comprehensive evaluation of the impact.

4-Aminopyridine Prevents the Conformational Changes Associated with P/C-Type Inactivation in Shaker Channels

The effect of 4-aminopyridine (4-AP) on Kv channel activation has been extensively investigated, but its interaction with inactivation is less well understood. Voltage-clamp fluorimetry was used to directly monitor the action of 4-AP on conformational changes associated with slow inactivation of Shaker channels. Tetramethylrhodamine-5-maleimide was used to fluorescently label substituted cysteine residues in the S3-S4 linker (A359C) and pore (S424C). Activation- and inactivation-induced changes in fluorophore microenvironment produced fast and slow phases of fluorescence that were modified by 4-AP. In Shaker A359C, 4-AP block reduced the slow-phase contribution from 61 ± 3 to 28 ± 5%, suggesting that binding inhibits the conformational changes associated with slow inactivation and increased the fast phase that reports channel activation from 39 ± 3 to 72 ± 5%. In addition, 4-AP enhanced both fast and slow phases of fluorescence return upon repolarization (τ reduced from 87 ± 15 to 40 ± 1 ms and from 739 ± 83 to 291 ± 21 ms, respectively), suggesting that deactivation and recovery from inactivation were enhanced. In addition, the effect of 4-AP on the slow phase of fluorescence was dramatically reduced in channels with either reduced (T449V) or permanent P-type (W434F) inactivation. Interestingly, the slow phase of fluorescence return of W434F channels was enhanced by 4-AP, suggesting that 4-AP prevents the transition to C-type inactivation in these channels. These data directly demonstrate that 4-AP prevents slow inactivation of Kv channels and that 4-AP can bind to P-type-inactivated channels and selectively inhibit the onset of C-type inactivation.

Separation of P/C- and U-type inactivation pathways in Kv1.5 potassium channels

P/C‐type inactivation of Kv channels is thought to involve conformational changes in the outer pore of the channel, culminating in a partial constriction of the selectivity filter. Recent studies have identified a number of phenotypic differences in the inactivation properties of different Kv channels, including different sensitivities to elevation of extracellular K+ concentration, and different state dependencies of inactivation. We have demonstrated that an alternatively spliced short form of Kv1.5, resulting in disruption of the T1 domain, exhibits a shift in the state dependence of inactivation in this channel, and in the current study we have examined this further to contrast the properties of inactivation from open versus closed states. In a TEA+‐sensitive mutant of Kv1.5 (Kv1.5 R487T), 10 mm extracellular TEA+ inhibits inactivation in both full‐length and T1‐deleted channels, but does not inhibit closed‐state inactivation in T1‐deleted channel forms. Similarly, substitution of K+ and Na+ with Cs+ ions in the recording medium inhibits inactivation of both full‐length and T1‐deleted channel forms, but fails to inhibit closed‐state inactivation of T1‐deleted channels. Collectively, these data distinguish between open‐state and closed‐state inactivation, and suggest the presence of multiple possible mechanisms of inactivation coexisting in Kv1 channels.

Voltage Clamp Fluorimetry Reveals a Novel Outer Pore Instability in a Mammalian Voltage-gated Potassium Channel

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K+ ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3–S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 ± 3% of the total signal and occurred with a τ of 3.4 ± 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K+ to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.

Reduced Kilovoltage in Computed Tomography–Guided Intervention in a Community Hospital: Effect on the Radiation Dose

Purpose The purpose of this study was to determine whether low-kilovoltage (80 or 100 kV) computed tomography (CT)-guided interventions performed in a community-based hospital are feasible and to compare radiation exposure incurred with conventional 120 kV potential. Materials and Methods Effective doses (ED) received by patients who underwent CT-guided intervention were analysed before and after a low-dose kilovoltage protocol was instituted in our department. We performed CT-guided procedures of 93 consecutive patients by using conventional 120-kV tube voltage (50 patients) and a low voltage of 80 or 100 kV for the remainder of this cohort. Automatic tube current modulation was enabled to obtain the best image quality. Procedure details were prospectively recorded and included examination site and type, slice width, tube voltage and current, dose length product, volume CT dose index, and size-specific dose estimate. Dose length product was converted to ED to account for radiosensitivity of specific organs. Statistical comparisons with test differences in the ED, volume CT dose index, size-specific dose estimate, and effective diameter (patient size) were made by using the Student t test. Results All but 6 of the procedures performed at 80 kV were successful, for a success rate of 86%. At lower voltages, the ED was significantly (P < .01) reduced, on average, by 57%, 73%, and 65% for the pelvic, chest, and abdomen procedures, respectively. Conclusion A low-dose radiation technique by using 80 or 100 kV results in a high technical success rate for pelvic, chest, and abdomen CT-guided interventional procedures, although dramatically decreasing radiation exposure. There was no significant difference in effective diameter (patient size) between the conventional and the low-dose groups, which would suggest that dose reduction was indeed a result of kVp change and not patient size.

Functional characterization of the LQT2-causing mutation R582C and the associated voltage-dependent fluorescence signal.

BACKGROUND The R582C mutation is one of many Long-QT Syndrome type 2 (LQT2)-causing mutations localized to the human ether-a-go-go related gene (hERG) channels S5-P linker subdomain, yet its specific mechanism of dysfunction has not been examined. OBJECTIVE This study sought to characterize the biophysical properties of the congenital LQT2-causing mutation, R582C, and utilize this mutation to provide the first report of voltage-dependent fluorescence from the S5-P linker. METHODS Properties of the R582C channels were characterized by heterologous expression in both HEK293 cells and Xenopus oocytes using a combination of patch-clamp, 2-electrode voltage-clamp, immunoblot assay, and voltage-clamp fluorimetry. RESULTS Expression of hERG R582C was found to be deficient in HEK293 cells, yet was amenable to rescue by incubation at reduced temperature or by treatment with dofetilide. Rescued channels expressed at levels comparable to wild type (WT) channels. Kinetic differences result in decreased outward repolarizing current evoked by an action potential clamp protocol. Voltage-clamp fluorimetry experiments utilized the introduced cysteine to covalently attach a fluorescent probe (tetramethylrhodamine-5-maleimide) to the S5-P linker to directly observe conformational changes occurring due to inactivation. CONCLUSION The major mechanism underlying pathogenicity of the R582C mutation is a trafficking deficiency, although channels also exhibit kinetic deficiencies, perhaps reflecting the position of the mutation in the pore turret. Voltage clamp fluorescence signals from R582C channels provide evidence that the hERG turret undergoes distinct conformational changes during inactivation.