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Effects of Temperature on Heteromeric Kv11.1a/1b and Kv11.3 Channels.

ABSTRACT: Kv11.1 channels are crucial in cardiac physiology, and there is increasing evidence of physiological roles of different Kv11 channels outside the heart. The HERG (human Kv11.1a) channel has previously been shown to carry substantially more current at elevated temperatures, and we have now comparably investigated the temperature dependence of neuronal Kv11.3 channels and the more ubiquitous heteromeric Kv11.1a/1b channels. Transiently expressed rat Kv11 channels were studied at 21°C, 30°C, and 35°C. At near-physiological temperature, the maximal sustained outward current density was almost three times the mean value obtained at room temperature for Kv11.1a/1b, and increased by ∼150% for Kv11.3. For both channels, reduced inactivation contributed to the current increase at higher temperature. Elevated temperature moved Kv11.1a/1b isochronal activation curves to more negative potentials, but shifted the potential of half-maximal Kv11.3 channel activation to more depolarized values and reduced its voltage sensitivity. Thus, increased temperature stabilized the open state over the closed state of Kv11.1a/1b channels and exerted the opposite effect on Kv11.3 channel activation. Both Kv11 channels exhibited an overall high temperature sensitivity of most gating parameters, with remarkably high Q10 factors of ∼5 for the rate of Kv11.1a/1b activation. The Q10 factors for Kv11.3 gating were more uniform, but still higher for activation than for inactivation kinetics. The results demonstrate that characteristic differences between Kv11.1a/1b and Kv11.3 determined at room temperature do not necessarily apply to physiological conditions. The data provided here can aid in the design of models that will enhance our understanding of the role of Kv11 currents in excitable cells.

SUBMITTER: Mauerhofer M

PROVIDER: S-EPMC4982939 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Heterologously expressed hERG channels represent a mainstay of in vitro drug safety screens intended to mitigate risk of cardiac IKr block and sudden cardiac death. This is true even as more channel types are adopted as part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) intended to elevate specificity and thus enhance throughput of promising lead drugs. Until now, hERG1a homomeric channels have been used as a proxy for IKr despite a wealth of evidence showing that hERG1a/1b heteromers better represent native channels in terms of protein abundance and channel biophysical and pharmacological properties. Past efforts to create a stable hERG1a/1b cell line were met with unpredictable silencing of hERG1b expression despite stable integration of the gene into the HEK293 cell genome. Here we report a new cell line stably expressing hERG1a, with hERG1b reliably controlled by an inducible promoter sensitive to doxycycline. Co-immunoprecipitation, Western blot analysis and patch-clamp electrophysiology confirm the heteromeric composition of the expressed channels. Association with hERG1b was found to promote hERG1a protein levels and enhance membrane current levels. Optimal conditions for drug screening and experimental investigation were achieved at 24 h exposure to 100 ng/ml doxycycline. Differences in pharmacological sensitivity between homomeric and heteromeric channels were observed for dofetilide and ebastine, but not fluoxetine, as evaluated by their IC50 values. Using these values in the O'Hara-Rudy-CiPA in silico model revealed discrepancies in pro-arrhythmia risk, implying the hERG1a homomeric platform overestimates risk for these two drugs. Dofetilide block was use-dependent and faster for hERG1a/1b than hERG1a channels, whereas ebastine showed considerable block at rest and had a slower progression for hERG1a/1b channels. The hERG1a/1b cell line thus represents an advanced model for contemporary drug safety screening assays such as CiPA that employ IC50 values to estimate risk of proarrhythmia in computational models of ventricular cardiomyocytes. This novel technology fulfills an unmet need to enhance specificity and foster a safe yet expanded drug development pipeline.

Project description:Silent voltage-gated potassium channel subunits (KVS) interact selectively with members of the KV2 channel family to modify their functional properties. The localization and functional roles of these silent subunits remain poorly understood. Mutations in the KVS subunit, KV8.2 (KCNV2), lead to severe visual impairment in humans, but the basis of these deficits remains unclear. Here, we examined the localization, native interactions, and functional properties of KV8.2-containing channels in mouse, macaque, and human photoreceptors of either sex. In human retina, KV8.2 colocalized with KV2.1 and KV2.2 in cone inner segments and with KV2.1 in rod inner segments. KV2.1 and KV2.2 could be coimmunoprecipitated with KV8.2 in retinal lysates indicating that these subunits likely interact directly. Retinal KV2.1 was less phosphorylated than cortical KV2.1, a difference expected to alter the biophysical properties of these channels. Using voltage-clamp recordings and pharmacology, we provide functional evidence for Kv2-containing channels in primate rods and cones. We propose that the presence of KV8.2, and low levels of KV2.1 phosphorylation shift the activation range of KV2 channels to align with the operating range of rod and cone photoreceptors. Our data indicate a role for KV2/KV8.2 channels in human photoreceptor function and suggest that the visual deficits in patients with KCNV2 mutations arise from inadequate resting activation of KV channels in rod and cone inner segments.SIGNIFICANCE STATEMENT Mutations in a voltage-gated potassium channel subunit, KV8.2, underlie a blinding inherited photoreceptor dystrophy, indicating an important role for these channels in human vision. Here, we have defined the localization and subunit interactions of KV8.2 channels in primate photoreceptors. We show that the KV8.2 subunit interacts with different Kv2 channels in rods and cones, giving rise to potassium currents with distinct functional properties. Our results provide a molecular basis for retinal dysfunction in patients with mutations in the KCNV2 gene encoding KV8.2.

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Effects of Temperature on Heteromeric Kv11.1a/1b and Kv11.3 Channels.

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