Recombinant Chicken Potassium voltage-gated channel subfamily H member 2 (KCNH2)

What is the structure and function of Chicken KCNH2?

Chicken KCNH2 (potassium voltage-gated channel subfamily H member 2) is a 526-amino acid protein that forms the alpha subunit of voltage-gated potassium channels responsible for regulating membrane potential. The full-length protein contains several functional domains including transmembrane segments, a pore region, and cytoplasmic domains that regulate channel gating. Its primary function is conducting potassium ions across cell membranes, particularly important in cardiac repolarization. The protein sequence contains critical motifs that determine voltage sensing, ion selectivity, and gating kinetics. Commercially available recombinant forms typically include an N-terminal His tag to facilitate purification and experimental manipulation . The protein shares significant homology with human KCNH2, which encodes the hERG channel responsible for the rapid component of cardiac delayed rectifier K+ current (IKr) . This conservation makes chicken KCNH2 valuable for comparative electrophysiological studies and structure-function analyses.

How should recombinant Chicken KCNH2 protein be stored and handled to maintain stability?

Recombinant Chicken KCNH2 protein requires specific storage conditions to maintain structural integrity and function. The lyophilized powder form offers greater stability for shipping and long-term storage compared to reconstituted protein . Upon receipt, the protein should be stored at -20°C to -80°C, with aliquoting strongly recommended to avoid detrimental freeze-thaw cycles. For reconstitution, deionized sterile water should be used to achieve concentrations between 0.1-1.0 mg/mL. The addition of glycerol at a final concentration of 5-50% significantly improves stability during storage, with 50% being optimal for long-term preservation . Working aliquots can be maintained at 4°C but should be used within one week to prevent degradation. The storage buffer composition (Tris/PBS-based buffer with 6% trehalose at pH 8.0) is specifically formulated to maintain protein stability . When handling the protein for experiments, minimize exposure to repeated temperature changes and avoid vortexing, which can cause protein denaturation. Instead, gentle mixing through inversion or slow pipetting is recommended to preserve the native conformation and functionality of the channel protein.

What are the key differences between chicken and human KCNH2 that researchers should consider?

While chicken and human KCNH2 share significant functional and structural homology, researchers must consider several key differences. The chicken variant is considerably shorter (526 amino acids) compared to human KCNH2 (approximately 1159 amino acids), primarily due to differences in regulatory domains . Despite this length disparity, the core channel-forming regions and primary functional domains show high conservation. Pharmacological responses may differ between species, with some compounds showing species-specific efficacy or potency. When using chicken KCNH2 as a model for human channel function, researchers should particularly note differences in temperature sensitivity, as avian body temperature (40-42°C) exceeds human physiological temperature. The chicken channel may exhibit altered kinetics and voltage-dependence compared to human KCNH2, necessitating careful interpretation when extrapolating findings between species. Additionally, interaction with accessory subunits and regulatory proteins may differ between chicken and human channels, potentially affecting channel modulation by signaling pathways. Depending on research questions, these differences can either be limitations to consider or advantageous features for comparative studies examining evolutionarily conserved channel properties across species.

What electrophysiological protocols are most effective for characterizing KCNH2 channel function?

Comprehensive characterization of KCNH2 channels requires specialized electrophysiological protocols that capture their unique biophysical properties. Patch-clamp electrophysiology remains the gold standard, with both manual and automated approaches providing complementary data . For activation properties, implement voltage-step protocols from a negative holding potential (-80mV) to a series of test potentials (-60 to +60mV in 10mV increments), followed by a repolarizing step to -50mV to elicit characteristic tail currents. This protocol reveals both the voltage-dependence of activation and deactivation kinetics. For inactivation properties, use a triple-pulse protocol: an initial depolarizing step to activate channels, a brief hyperpolarizing step to recover channels from inactivation, followed by test pulses to various potentials to measure the voltage-dependence of inactivation. Temperature control is crucial as KCNH2 kinetics are highly temperature-dependent; ideally, recordings should be performed at physiological temperature (37°C) rather than room temperature. For pharmacological studies, implement step-pulse protocols repeated at regular intervals (15-30 seconds) during drug application to assess both tonic block and use-dependent effects. Recovery from inactivation can be measured using a double-pulse protocol with variable interpulse intervals. Finally, action potential clamp protocols using prerecorded cardiac action potentials provide physiologically relevant assessment of channel function throughout the cardiac cycle.

How can researchers design effective shRNAs for KCNH2 suppression in gene therapy approaches?

Designing effective shRNAs for KCNH2 suppression requires a systematic approach to ensure potency, specificity, and compatibility with gene therapy applications. Begin by identifying target sequences within KCNH2 mRNA that are conserved across relevant splice variants but devoid of common genetic polymorphisms that could affect knockdown efficiency . Bioinformatic screening should eliminate sequences with significant homology to other genes, particularly related potassium channels, to minimize off-target effects. Design multiple candidate shRNAs (typically 5-10) targeting different regions of the mRNA to identify the most effective constructs. For validation, co-transfect each shRNA with wild-type KCNH2 in appropriate cell lines (e.g., TSA201) and assess knockdown efficiency using both qRT-PCR and western blot analysis, with effective candidates demonstrating at least 70-80% reduction in expression . Test selected candidates against known KCNH2 variants to ensure variant-independent suppression, particularly important for therapeutic applications targeting diverse patient mutations. For suppression-replacement strategies, design an shRNA-immune replacement gene by introducing synonymous mutations at the wobble positions within the shRNA target sequence, creating resistance to the shRNA while maintaining the amino acid sequence . This approach enables simultaneous suppression of endogenous (potentially mutant) KCNH2 while providing functional replacement with the modified wild-type channel. Finally, demonstrate dose-dependent effects of the shRNA to establish the therapeutic window between efficacy and potential toxicity.

What approaches should be used to study trafficking defects in KCNH2 mutants?

Studying trafficking defects in KCNH2 mutants requires a multi-faceted approach combining imaging, biochemical, and functional techniques. Begin with fluorescently tagged KCNH2 constructs (wild-type and mutant) expressed in appropriate cell lines and analyze subcellular localization using confocal microscopy with markers for relevant compartments (plasma membrane, endoplasmic reticulum, Golgi apparatus, and lysosomes). This allows visualization of where mutant channels are retained in the secretory pathway . Complement imaging with biochemical analysis using western blotting to detect glycosylation states—KCNH2 produces two bands representing immature core-glycosylated (135 kDa) and mature complex-glycosylated (155 kDa) forms, with trafficking-defective mutants showing reduced or absent mature form . Implement surface biotinylation assays to quantitatively measure membrane expression levels, providing a more precise measurement than microscopy alone. For functional confirmation, conduct patch-clamp electrophysiology to correlate trafficking defects with current density reduction. Temperature-dependent rescue experiments provide valuable mechanistic insights—incubation at reduced temperature (27°C) often rescues trafficking-defective but not functionally defective mutants . Pharmacological approaches using known trafficking correctors (e.g., E-4031, fexofenadine, lumacaftor) can identify whether defects are amenable to chemical chaperone therapy. For comprehensive analysis, implement pulse-chase experiments with metabolic labeling to track the time course of protein maturation and degradation, revealing whether defects involve accelerated degradation or impaired forward trafficking.

What methods are most effective for studying interactions between KCNH2 and potential therapeutic compounds?

Studying interactions between KCNH2 and therapeutic compounds requires a comprehensive approach encompassing binding, functional effects, and therapeutic potential. Begin with in silico screening using molecular docking against homology models or crystal structures to identify potential binding sites and candidate compounds. For direct binding assessment, implement radioligand binding assays using labeled reference compounds (e.g., [3H]dofetilide) with competitive displacement to determine binding affinity constants for novel compounds. Surface plasmon resonance provides an alternative label-free approach to study binding kinetics in real-time. Functional effects should be assessed using voltage-clamp electrophysiology with protocols that explore state-dependent interactions—many compounds preferentially bind to open or inactivated channel states . Implement voltage protocols that isolate specific gating transitions (activation, inactivation, recovery) to identify precise mechanistic effects of compounds. For high-throughput initial screening, automated patch-clamp platforms or fluorescence-based potentiometric dyes can evaluate large compound libraries. Temperature effects are critical to consider, as many KCNH2-compound interactions are highly temperature-dependent. For therapeutic evaluation, assess both acute effects (direct channel modulation) and chronic effects (potential influence on trafficking) through prolonged incubation studies. Implement concentration-response curves to determine potency (EC50/IC50) and efficacy parameters. For potential rescue compounds targeting trafficking-defective mutants, combine electrophysiology with biochemical trafficking assays to confirm both improved membrane expression and functional correction. Finally, validate promising compounds in more physiological systems such as cardiomyocytes to confirm efficacy in native cellular environments.

What controls are essential when studying KCNH2 function and modulation?

Robust experimental design for KCNH2 studies requires comprehensive controls to ensure valid interpretations and reproducible results. For expression studies, include both positive controls (wild-type KCNH2 under identical conditions) and negative controls (untransfected cells and empty vector transfections) to establish baseline channel function and account for endogenous currents . When studying heterozygous conditions, implement co-expression of wild-type and mutant channels in a 1:1 ratio to mimic the clinical scenario, alongside homozygous wild-type and homozygous mutant expressions for comparison. For pharmacological interventions, include vehicle controls subjected to identical solution exchanges and known reference compounds with established effects on KCNH2 (e.g., E-4031 as a specific blocker). In electrophysiological experiments, implement time-matched controls to account for potential current rundown or changes in channel properties over recording duration. For trafficking studies, include both a known trafficking-defective KCNH2 mutant as a positive control and a non-KCNH2 membrane protein as a control for general trafficking machinery function . In RNA interference experiments, non-targeting shRNA controls are essential to distinguish specific suppression from non-specific effects of the expression system . Temperature sensitivity experiments should include both the experimental and control constructs at all tested temperatures. When assessing mutation effects, conservative substitutions at the same position can help distinguish effects due to specific amino acid properties versus general structural disruption. These comprehensive controls collectively ensure that observed phenotypes can be specifically attributed to the experimental manipulation under investigation.

How should researchers design experiments to integrate structural, functional, and clinical data for KCNH2?

Integrating structural, functional, and clinical data for KCNH2 requires thoughtful experimental design that bridges these distinct domains. Begin by selecting mutations or compounds for study based on their location within the channel structure, particularly targeting key functional domains like the voltage sensor, pore region, or C-terminal regulatory domains . Implement systematic mutagenesis of specific structural elements followed by parallel assessment via electrophysiology (functional impact), biochemistry (expression, trafficking), and computational modeling (structural perturbations). Correlate functional parameters (activation V₁/₂, inactivation kinetics) with structural features (residue accessibility, domain interactions) to establish structure-function relationships. For clinical correlation, classify functional defects according to severity (mild, moderate, severe) based on quantitative parameters like current density reduction, kinetic abnormalities, or trafficking deficiency, then correlate these classifications with clinical phenotypes from patient databases . Implement cardiac action potential modeling to translate channel-level effects to cellular electrophysiology, predicting how specific functional changes might alter cardiac repolarization and arrhythmia susceptibility. For comprehensive analysis of mutations, examine both heterozygous (wild-type + mutant) and homozygous (mutant only) conditions to assess dominant-negative effects relevant to the clinical heterozygous state. When studying potential therapeutic compounds, characterize effects across multiple parameters (activation, inactivation, deactivation, trafficking) to develop a comprehensive functional profile. Finally, establish standardized severity classifications that integrate multiple functional parameters to better predict clinical outcomes and therapeutic responses, moving toward a more quantitative framework for translating KCNH2 functional data to clinical significance.

What are the key considerations when comparing results between different expression systems for KCNH2?

When comparing KCNH2 results between different expression systems, researchers must account for several system-dependent variables that can significantly impact channel behavior. First, consider the intrinsic differences in cellular machinery—mammalian systems (HEK293, CHO) provide appropriate post-translational modifications and trafficking pathways for KCNH2, while bacterial systems (E. coli) are useful only for protein production and biochemical studies, not functional assessments . Expression levels vary dramatically between systems and affect channel behavior; excessive overexpression can lead to non-physiological current properties and potential retention in intracellular compartments. The presence of endogenous currents differs between cell types—CHO cells generally exhibit fewer interfering currents than HEK293 cells, affecting background subtraction needs and signal-to-noise ratios. Membrane composition varies between expression systems, potentially affecting channel function through differences in membrane fluidity, cholesterol content, and lipid rafts. Temperature sensitivity is another critical factor—many studies in heterologous systems are conducted at room temperature, while native channels function at 37°C with significantly different kinetics. The absence of cardiac-specific accessory proteins in heterologous systems may result in channel behavior that differs from native cardiomyocytes; consider co-expression with relevant partners (minK, MiRP1) for more physiologically relevant data. For accurate comparison between systems, standardize recording conditions including ionic composition, temperature, expression duration, and voltage protocols. Finally, when extrapolating from heterologous systems to clinical significance, acknowledge that channel behavior in artificial systems represents only an approximation of in vivo function and should be interpreted with appropriate caution.

How can researchers address poor expression or non-functional KCNH2 in experimental systems?

Poor expression or non-functional KCNH2 represents a common challenge requiring systematic troubleshooting approaches. For bacterial expression systems, optimize conditions by employing specialized strains (Rosetta, BL21) that supply rare tRNAs, reducing induction temperature (16-20°C), and using milder induction conditions to enhance proper folding . For mammalian expression, evaluate transfection efficiency using co-expressed fluorescent markers and optimize transfection parameters including DNA quality, cell confluence (70-80% typically optimal), and transfection reagent ratios. If protein toxicity is suspected, implement inducible expression systems that allow controlled expression levels. Address potential RNA stability issues by checking for cryptic splice sites or destabilizing elements in the construct design. For trafficking-deficient constructs, incubation at reduced temperature (27-30°C) can promote proper folding and membrane expression. Consider co-expression with chaperone proteins (Hsp70, Hsp90) that facilitate ion channel folding and trafficking. For functional studies, ensure recording solutions contain appropriate K+ concentrations, as KCNH2 function is highly sensitive to external potassium levels. If channel inactivation is too rapid to measure currents effectively, implement protocols using elevated external K+ (20-40 mM) which reduces inactivation and enhances current amplitude. For purified protein applications, screen multiple detergents and lipid environments to identify optimal solubilization conditions. When mutations affect channel function, create rescue constructs by introducing compensatory mutations based on structure-function understanding or by adding trafficking-enhancement tags. Finally, for difficult-to-express constructs, consider alternative expression strategies including baculovirus-insect cell systems which often provide higher expression of functional membrane proteins than mammalian or bacterial systems.

What approaches can distinguish between trafficking and functional defects in KCNH2 mutants?

Distinguishing between trafficking and functional defects in KCNH2 mutants requires a strategic combination of complementary techniques that probe different aspects of channel biology. Begin with Western blot analysis to assess the glycosylation state of the channel protein—trafficking-deficient mutants typically show reduced levels of the mature, complex-glycosylated form (155 kDa) relative to the immature core-glycosylated form (135 kDa) . Complement this with confocal microscopy using fluorescently tagged constructs co-stained with markers for cellular compartments to visualize channel location; trafficking-deficient channels show co-localization with ER or Golgi markers rather than membrane localization. Surface biotinylation provides quantitative measurement of membrane expression, allowing direct comparison between wild-type and mutant channels. For functional assessment, implement patch-clamp electrophysiology with analysis of both current density (indicating channel number) and kinetic properties (reflecting intrinsic channel function). The definitive approach for distinguishing these defects involves rescue experiments—incubation at reduced temperature (27°C) or with pharmacological chaperones typically rescues trafficking but not functional defects . Calculate the ratio of current density to surface expression; a reduced ratio in a mutant with normal surface expression indicates a functional defect with normal trafficking. For mixed defects (both trafficking and functional abnormalities), implement single-channel recordings to determine whether channels that reach the membrane have normal conductance and gating. Finally, rate-limiting step analysis using pulse-chase experiments can identify whether defects involve accelerated degradation or impaired forward trafficking, providing further mechanistic insight into the nature of the defect.

How can researchers troubleshoot inconsistent electrophysiological recordings of KCNH2?

Inconsistent electrophysiological recordings of KCNH2 channels can stem from multiple sources requiring systematic troubleshooting approaches. First, standardize cell culture conditions, as variability in passage number, confluence, and time post-transfection significantly impacts channel expression and function. Implement stringent quality control for patch-clamp experiments including consistent seal resistance criteria (>1 GΩ), continuous access resistance monitoring, and complete capacity compensation. KCNH2 is particularly temperature-sensitive—maintain consistent temperature control using heated perfusion systems and monitor actual bath temperature rather than controller settings. Address solution-related variables by preparing fresh solutions regularly, controlling for pH drift, and ensuring consistent osmolarity. KCNH2 currents are notably sensitive to extracellular K+ concentration—standardize this parameter and account for it when comparing datasets . Implement consistent voltage protocols with sufficient recovery time between sweeps (30-60 seconds) to allow complete channel recovery from inactivation. For pharmacological studies, account for compound adsorption to perfusion tubing by using inert materials and pre-saturating the system. When recording from transiently transfected cells, select cells with moderate fluorescence intensity, as extreme overexpression can lead to abnormal channel behavior. Implement data quality filters during analysis, excluding cells with excessive rundown or unstable holding currents. Additionally, standardize analysis parameters including leak subtraction methods, current measurement points, and fitting procedures for activation and inactivation curves. Track and report environmental factors such as room temperature fluctuations and electromagnetic noise that may impact sensitive KCNH2 recordings. Finally, implement blind analysis when possible, with the experimenter unaware of the experimental condition to minimize unconscious bias in cell selection or analysis.

What strategies help differentiate KCNH2 currents from endogenous currents in expression systems?

Distinguishing KCNH2 currents from endogenous currents requires strategic experimental design and analytical approaches tailored to the channel's unique properties. First, thoroughly characterize the endogenous current profile of your chosen expression system through detailed voltage-clamp recordings of untransfected cells under identical conditions . Select expression systems with minimal interfering currents—CHO cells often provide cleaner backgrounds than HEK293 cells for KCNH2 studies. Implement KCNH2-specific voltage protocols that exploit its distinctive biophysical signature, particularly the characteristic bell-shaped current-voltage relationship resulting from rapid inactivation at positive potentials and prominent tail currents upon repolarization. Apply pharmacological isolation using specific KCNH2 blockers (E-4031, dofetilide at 1-5 μM) to isolate KCNH2 current by subtraction of pre- and post-drug currents. Incorporate internal cesium in the recording pipette solution to block many endogenous potassium currents while preserving KCNH2 function. Express KCNH2 with fluorescent protein tags to select only cells with verified expression for recording, ensuring signal originates from transfected channels. For mathematical isolation of overlapping currents, apply biophysical modeling with multi-component fitting of activation and inactivation kinetics. When using inducible expression systems, perform paired recordings before and after KCNH2 induction in the same cell, using the pre-induction recording as the perfect control for endogenous currents. For comprehensive validation, combine electrophysiological identification with biochemical verification through Western blot or surface expression assays to confirm that observed currents correlate with KCNH2 protein expression levels.

What statistical approaches are most appropriate for analyzing electrophysiological data from KCNH2 studies?

Analyzing electrophysiological data from KCNH2 studies requires statistical approaches tailored to the complex, multiparametric nature of channel function. For current-voltage relationships, implement repeated measures ANOVA when comparing multiple experimental groups across voltage points, followed by appropriate post-hoc tests (Tukey or Bonferroni) to control for multiple comparisons . For activation and inactivation curves, first fit individual cell data to Boltzmann functions to extract parameters (V₁/₂, slope factor), then compare these parameters across groups using parametric (t-test, ANOVA) or non-parametric tests depending on data distribution. When analyzing kinetic parameters (activation, deactivation, inactivation time constants), apply logarithmic transformation before statistical testing as these values often follow log-normal rather than normal distributions. For drug response studies, fit concentration-response data to Hill equations to derive IC₅₀/EC₅₀ values and Hill coefficients, then compare these parameters using extra sum-of-squares F tests. When comparing mutation effects across multiple parameters, consider multivariate approaches such as principal component analysis to identify patterns of functional changes that may not be apparent in univariate analyses. For time-series data such as current rundown or drug washout, apply mixed-effects models that account for both fixed effects (treatment, genotype) and random effects (cell-to-cell variability). Implement robust power analyses during experimental design, recognizing that electrophysiological data often exhibits high variability requiring larger sample sizes than typically used in molecular biology. Report not only significance values but also effect sizes and confidence intervals to provide a more complete statistical picture of KCNH2 functional changes.

How should researchers interpret the clinical significance of KCNH2 functional data?

Interpreting the clinical significance of KCNH2 functional data requires a translational framework connecting molecular findings to potential pathophysiological consequences. Establish standardized severity classifications for functional defects based on current density reduction, kinetic alterations, and trafficking abnormalities, correlating these with clinical phenotype databases . Implement computational modeling using cardiac action potential simulations to translate channel-level changes to cellular electrophysiology—incorporate experimental data into established models to predict action potential duration, repolarization abnormalities, and arrhythmia triggers. For heterozygous conditions typical in clinical settings, focus on experiments with defined wild-type:mutant ratios (50:50) that mimic patient genotypes. Develop quantitative metrics linking channel defects to arrhythmia risk through integration of multiple parameters rather than single metrics—principal component analysis can help identify patterns of channel dysfunction that best predict clinical severity. Consider physiological modulators—experiment under conditions mimicking sympathetic stimulation (isoproterenol, elevated temperature) and other relevant stressors (hypokalemia, acidosis) that may unveil latent channel dysfunction. For therapeutic implications, systematically evaluate pharmacological responses of mutant channels to establish mutation-specific drug efficacy and risk profiles. Implement population-level analysis by studying multiple variants affecting the same structural domain to develop domain-specific clinical correlations. For precision medicine applications, develop computational models incorporating patient-specific factors alongside channel data to provide individualized risk assessment. Finally, validate functional severity classifications through correlation with available clinical data, establishing whether in vitro parameters predict clinical outcomes.

What approaches should be used to analyze data from gene therapy strategies targeting KCNH2?

Analyzing data from gene therapy approaches targeting KCNH2 requires specialized frameworks addressing both mechanistic efficacy and therapeutic potential. For suppression-replacement strategies, implement quantitative analysis of three key components: suppression efficiency (percentage reduction of endogenous/mutant KCNH2), replacement expression (absolute and relative to endogenous levels), and functional restoration (percentage of wild-type current density and kinetic normalization) . Establish dose-response relationships for the therapeutic construct using multiple doses/multiplicities of infection to determine the therapeutic window between minimal effective dose and potential toxicity thresholds. For longitudinal assessment, develop time-course analyses tracking expression and function from initial delivery through steady-state and potential decline phases. Implement comprehensive off-target analysis including transcriptome profiling to detect unintended suppression of related genes, particularly other potassium channel family members. When assessing variant-independent approaches, systematically evaluate efficacy across multiple mutation types (trafficking-deficient, kinetically abnormal, dominant-negative) to establish broad applicability . For in vivo evaluations, develop multi-scale analysis correlating molecular correction (mRNA/protein levels) with cellular electrophysiology (patch-clamp), tissue-level function (ex vivo optical mapping), and organism-level outcomes (ECG parameters, arrhythmia susceptibility). Implement rigorous statistical approaches including mixed-effects models that account for both treatment effects and subject-to-subject variability. For translational potential, develop predictive algorithms linking molecular correction thresholds to anticipated clinical benefit, establishing the minimum correction required for meaningful phenotype rescue. Finally, implement integrated safety assessment including immune response evaluation, potential proarrhythmic effects from heterogeneous expression, and long-term studies for insertional mutagenesis or expression drift.

How can researchers effectively compare data across different experimental models of KCNH2?

Effective comparison of KCNH2 data across experimental models requires standardized approaches that account for model-specific variables. Begin by establishing common experimental conditions across models—consistent recording solutions, temperature, and voltage protocols provide the foundation for valid comparisons . Implement normalization strategies appropriate to the experimental question; for current density comparisons, normalize to cell capacitance, while for kinetic parameters, temperature correction using Q10 values can adjust for temperature differences between recording conditions. When comparing heterologous expression to native systems, account for differences in channel density by normalizing to maximum current or considering relative changes rather than absolute values. Develop standardized reporting formats that include all relevant experimental parameters (expression system, recording solutions, temperature, time post-transfection, voltage protocols) to facilitate comparison across laboratories. For pharmacological studies, express drug effects as percentage change from baseline rather than absolute values to account for model-dependent differences in baseline current. When comparing mutant effects across models, calculate the relative change compared to wild-type in the same model rather than comparing absolute values between models. For complex comparisons involving multiple parameters, implement multivariate statistical approaches including principal component analysis or clustering algorithms to identify patterns that may not be apparent in individual parameter comparisons. Validate key findings in multiple models whenever possible, as convergent results across different systems provide stronger evidence than findings from a single model. Finally, acknowledge model-specific limitations in your interpretation, recognizing that each experimental system provides a different perspective on channel function rather than an absolute "truth" about KCNH2 behavior in vivo.