Gastrointestinal Symptoms and Channelopathy-Associated Epilepsy.

OBJECTIVE: To determine the prevalence of and identify factors associated with gastrointestinal (GI) symptoms among children with channelopathy-associated developmental and epileptic encephalopathy (DEE). STUDY DESIGN: Parents of 168 children with DEEs linked to SCN1A (n = 59), KCNB1 (n = 31), or KCNQ2 (n = 78) completed online CLIRINX surveys about their children's GI symptoms. Our analysis examined the prevalence, frequency, and severity of GI symptoms, as well as DEE type, functional mobility, feeding difficulties, ketogenic diet, antiseizure medication, autism spectrum disorder (ASD), and seizures. Statistical analyses included the χ2 test, Wilcoxon rank-sum analysis, and multiple logistic regression. RESULTS: GI symptoms were reported in 92 of 168 patients (55%), among whom 63 of 86 (73%) reported daily or weekly symptoms, 29 of 92 (32%) had frequent or serious discomfort, and 13 of 91 (14%) had frequent or serious appetite disturbances as a result. The prevalence of GI symptoms varied across DEE cohorts with 44% of SCN1A-DEE patients, 35% of KCNB1-DEE patients, and 71% of KCNQ2-DEE patients reporting GI symptoms in the previous month. After adjustment for DEE type, current use of ketogenic diet (6% reported), and gastrostomy tube (13% reported) were both associated with GI symptoms in a statistically, but not clinically, significant manner (P < .05). Patient age, functional mobility, feeding difficulties, ASD, and seizures were not clearly associated with GI symptoms. Overall, no individual antiseizure medication was significantly associated with GI symptoms across all DEE cohorts. CONCLUSIONS: GI symptoms are common and frequently severe in patients with DEE.

Cytoskeleton disruption affects Kv2.1 channel function and its modulation by PIP2.

Voltage-gated potassium channels are expressed in a wide variety of excitable and non-excitable cells and regulate numerous cellular functions. The activity of ion channels can be modulated by direct interaction or/and functional coupling with other proteins including auxiliary subunits, scaffold proteins and the cytoskeleton. Here, we evaluated the influence of the actin-based cytoskeleton on the Kv2.1 channel using pharmacological and electrophysiological methods. We found that disruption of the actin-based cytoskeleton by latrunculin B resulted in the regulation of the Kv2.1 inactivation mechanism; it shifted the voltage of half-maximal inactivation toward negative potentials by approximately 15 mV, accelerated the rate of closed-state inactivation, and delayed the recovery rate from inactivation. The actin cytoskeleton stabilizing agent phalloidin prevented the hyperpolarizing shift in the half-maximal inactivation potential when co-applied with latrunculin B. Additionally, PIP2 depletion (a strategy that regulates Kv2.1 inactivation) after cytoskeleton disruption does not regulate further the inactivation of Kv2.1, which suggests that both factors could be regulating the Kv2.1 channel by a common mechanism. In summary, our results suggest a role for the actin-based cytoskeleton in regulating Kv2.1 channels.

The Insensitivity of TASK-3 K2P Channels to External Tetraethylammonium (TEA) Partially Depends on the Cap Structure.

Two-pore domain K⁺ channels (K2P) display a characteristic extracellular cap structure formed by two M1-P1 linkers, the functional role of which is poorly understood. It has been proposed that the presence of the cap explains the insensitivity of K2P channels to several K⁺ channel blockers including tetraethylammonium (TEA). We have explored this hypothesis using mutagenesis and functional analysis, followed by molecular simulations. Our results show that the deletion of the cap structure of TASK-3 (TWIK-related acid-sensitive K⁺ channel) generates a TEA-sensitive channel with an IC50 of 11.8 ± 0.4 mM. The enhanced sensitivity to TEA displayed by the cap-less channel is also explained by the presence of an extra tyrosine residue at position 99. These results were corroborated by molecular simulation analysis, which shows an increased stability in the binding of TEA to the cap-less channel when a ring of four tyrosine is present at the external entrance of the permeation pathway. Consistently, Y99A or Y205A single-residue mutants generated in a cap-less channel backbone resulted in TASK-3 channels with low affinity to external TEA.

Regulation of Kv2.1 channel inactivation by phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane phospholipid that regulates the function of multiple ion channels, including some members of the voltage-gated potassium (Kv) channel superfamily. The PIP2 sensitivity of Kv channels is well established for all five members of the Kv7 family and for Kv1.2 channels; however, regulation of other Kv channels by PIP2 remains unclear. Here, we investigate the effects of PIP2 on Kv2.1 channels by applying exogenous PIP2 to the cytoplasmic face of excised membrane patches, activating muscarinic receptors (M1R), or depleting endogenous PIP2 using a rapamycin-translocated 5-phosphatase (FKBP-Inp54p). Exogenous PIP2 rescued Kv2.1 channels from rundown and partially prevented the shift in the voltage-dependence of inactivation observed in inside-out patch recordings. Native PIP2 depletion by the recruitment of FKBP-Insp54P or M1R activation in whole-cell experiments, induced a shift in the voltage-dependence of inactivation, an acceleration of the closed-state inactivation, and a delayed recovery of channels from inactivation. No significant effects were observed on the activation mechanism by any of these treatments. Our data can be modeled by a 13-state allosteric model that takes into account that PIP2 depletion facilitates inactivation of Kv2.1. We propose that PIP2 regulates Kv2.1 channels by interfering with the inactivation mechanism.

Phytochemicals genistein and capsaicin modulate Kv2.1 channel gating.

BACKGROUND: Phytochemicals are a large group of plant-derived compounds that have a broad range of pharmacological effects. Some of these effects are derived from their action on transport proteins, including ion channels. The present study investigates the effects of the phytochemicals genistein and capsaicin on voltage-gated potassium Kv2.1 channels. METHODS: The whole-cell patch clamp technique was used to explore the regulation of Kv2.1 channels expressed in HEK293 cells by genistein and capsaicin. RESULTS: Both phytochemicals had a profound effect on the gating properties of Kv2.1 channels; the voltage dependence of activation and inactivation was shifted to hyperpolarized potentials, the closed-state inactivation was accelerated, and the recovery from inactivation was delayed. Moreover, genistein and capsaicin inhibited Kv2.1 currents in a concentration dependent manner. CONCLUSION: This study effectively demonstrated the inhibitory effects of genistein and capsaicin on Kv2.1 channels. As Kv2.1 channels play a prominent role in glucose-stimulated insulin secretion, our findings contribute to our understanding of the putative mechanism by which these phytochemicals exert their reported hypoglycemic effects.

Immunosuppressive evidence of Tityus serrulatus toxins Ts6 and Ts15: insights of a novel K(+) channel pattern in T cells.

The voltage-gated potassium channel Kv1.3 is a novel target for immunomodulation of autoreactive effector memory T cells, which play a major role in the pathogenesis of autoimmune diseases. In this study, the Ts6 and Ts15 toxins isolated from Tityus serrulatus (Ts) were investigated for their immunosuppressant roles on CD4(+) cell subsets: naive, effector (TEF ), central memory (TCM) and effector memory (TEM). The electrophysiological assays confirmed that both toxins were able to block Kv1.3 channels. Interestingly, an extended Kv channel screening shows that Ts15 blocks Kv2.1 channels. Ts6 and Ts15 significantly inhibit the proliferation of TEM cells and interferon-γ production; however, Ts15 also inhibits other CD4(+) cell subsets (naive, TEF and TCM). Based on the Ts15 inhibitory effect of proliferation of all CD4(+) cell subsets, and based on its blocking effect on Kv2.1, we investigated the Kv2.1 expression in T cells. The assays showed that CD4(+) and CD8(+) cells express the Kv2.1 channels mainly extracellularly with TCM cells expressing the highest number of Kv2.1 channels. We also provide in vivo experimental evidence to the protective effect of Ts6 and Ts15 on delayed-type hypersensitivity reaction. Altogether, this study presents the immunosuppressive behaviour of Ts6 and Ts15 toxins, indicating that these toxins could be promising candidates for autoimmune disease therapy. Moreover, this is the first report illustrating the involvement of a novel K(+) channel subtype, Kv2.1, and its distribution in T-cell subsets.

Modulation of Kv2.1 channels inactivation by curcumin.

BACKGROUND: The aim of the present study was to assess the effects of curcumin on the voltage-dependent Kv2.1 potassium channel. METHODS: The whole-cell patch-clamp technique was used to explore the regulation of Kv2.1 channels expressed in HEK293 cells by curcumin. RESULTS: Curcumin reduced the Kv2.1 currents; the inhibition occurred with a slow time course and was partially reversible. Curcumin did not alter the kinetics and voltage dependence of activation; however, the kinetics of open- and closed-state inactivation was accelerated by curcumin along with a hyperpolarizing shift in the voltage dependence of inactivation. Curcumin inhibition of Kv2.1 current was not use-dependent. CONCLUSIONS: Overall, our data suggest that curcumin inhibits Kv2.1 channels by modulating the inactivation gating, which would be expected to impact cellular physiology.

Quinidine interaction with Shab K+ channels: pore block and irreversible collapse of the K+ conductance.

Quinidine is a commonly used antiarrhythmic agent and a tool to study ion channels. Here it is reported that quinidine equilibrates within seconds across the Sf9 plasma membrane, blocking the open pore of Shab channels from the intracellular side of the membrane in a voltage-dependent manner with 1:1 stoichiometry. On binding to the channels, quinidine interacts with pore K(+) ions in a mutually destabilizing manner. As a result, when the channels are blocked by quinidine with the cell bathed in an external medium lacking K(+), the Shab conductance G(K) collapses irreversibly, despite the presence of a physiological [K(+)] in the intracellular solution. The quinidine-promoted collapse of Shab G(K) resembles the collapse of Shaker G(K) observed with 0 K(+) solutions on both sides of the membrane: thus the extent of G(K) drop depends on the number of activating pulses applied in the presence of quinidine, but is independent of the pulse duration. Taken together the observations indicate that, as in Shaker, the quinidine-promoted collapse of Shab G(K) occurs during deactivation of the channels, at the end of each activating pulse, with a probability of 0.1 per pulse at 80 mV. It appears that when Shab channels are open, the pore conformation able to conduct is stable in the absence of K(+), but on deactivation this conformation collapses irreversibly.

Stability of the Shab K+ channel conductance in 0 K+ solutions: the role of the membrane potential.

Shab channels are fairly stable with K(+) present on only one side of the membrane. However, on exposure to 0 K(+) solutions on both sides of the membrane, the Shab K(+) conductance (G(K)) irreversibly drops while the channels are maintained undisturbed at the holding potential. Herein it is reported that the drop of G(K) follows first-order kinetics, with a voltage-dependent decay rate r. Hyperpolarized potentials drastically inhibit the drop of G(K). The G(K) drop at negative potentials cannot be explained by a shift in the voltage dependence of activation. At depolarized potentials, where the channels undergo a slow inactivation process, G(K) drops in 0 K(+) with rates slower than those predicted based on the behavior of r at negative potentials, endowing the r-V(m) relationship with a maximum. Regardless of voltage, r is very small compared with the rate of ion permeation. Observations support the hypothesized presence of a stabilizing K(+) site (or sites) located either within the pore itself or in its external vestibule, at an inactivation-sensitive location. It is argued that part of the G(K) stabilization achieved at hyperpolarized potentials could be the result of a conformational change in the pore itself.