Evidence for the interaction of Endophilin A3 with endogenous K(Ca)2.3 channels in PC12 cells

Background/Aims: Small-conductance calcium-activated (SK) channels play an important role by controlling the after-hyperpolarization of excitable cells. The level of expression and density of these channels is an essential factor for controlling different cellular functions. Several studies showed a co-localization of KCa2.3 channels and Endophilin A3 in different tissues. Endophilin A3 belongs to a family of BAR- and SH3 domain containing proteins that bind to dynamin and are involved in the process of vesicle scission in clathrin-mediated endocytosis. Methods: Using the yeast two-hybrid system and the GST pull down assay we demonstrated that Endophilin A3 interacts with the N-terminal part of KCa2.3 channels. In addition, we studied the impact of this interaction on channel activity by patch clamp measurements in PC12 cells expressing endogenous KCa2.3 channels. KCa2.3 currents were activated by using pipette solutions containing 1 µM free Ca(2+). Results: Whole-cell measurements of PC12 cells transfected with Endophilin A3 showed a reduction of KCa2.3 specifc Cs(+) currents indicating that the interaction of Endophilin A3 with KCa2.3 channels also occurs in mammalian cells and that this interaction has functional consequences for current flowing through KCa2.3 channels. Since KCa2.3 specific currents could be increased in PC12 cells transfected with Endophilin A3 with DC-EBIO (30 µM), a known SK-channel activator, these data also implicate that Endophilin A3 did not significantly remove KCa2.3 channels from the membrane but changed the sensitivity of the channels to Ca(2+) which could be overcome by DC-EBIO. Conclusion: This interaction seems to be important for the function of KCa2.3 channels and might therefore play a significant role in situations where channel activation is pivotal for cellular function

Interaction of d-Tubocurarine with Potassium Channels: Molecular Modeling and Ligand Binding

ABSTRACT Potassium channels play fundamental roles in physiology. Chemically diverse drugs bind in the pore region of K ϩ channels. Here, we homology-modeled voltage-and Ca 2ϩ -gated

Voltage-gated potassium channels (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The 6TM family of K channels comprises the voltage-gated KV subfamilies, the EAG subfamily (which includes hERG channels), the Ca2+-activated Slo subfamily (actually with 7TM, termed BK) and the Ca2+-activated SK subfamily. These channels possess a pore-forming α subunit that comprise tetramers of identical subunits (homomeric) or of different subunits (heteromeric). Heteromeric channels can only be formed within subfamilies (e.g. Kv1.1 with Kv1.2; Kv7.2 with Kv7.3). The pharmacology largely reflects the subunit composition of the functional channel

Voltage-gated potassium channels (Kv) in GtoPdb v.2021.3

The 6TM family of K channels comprises the voltage-gated KV subfamilies, the EAG subfamily (which includes hERG channels), the Ca2+-activated Slo subfamily (actually with 7TM, termed BK) and the Ca2+-activated SK subfamily. These channels possess a pore-forming α subunit that comprise tetramers of identical subunits (homomeric) or of different subunits (heteromeric). Heteromeric channels can only be formed within subfamilies (e.g. Kv1.1 with Kv1.2; Kv7.2 with Kv7.3). The pharmacology largely reflects the subunit composition of the functional channel

Voltage-dependent inactivation gating at the selectivity filter of the MthK K+ channel

Voltage-dependent K+ channels can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel’s selectivity filter. C-type inactivation may involve movements of transmembrane voltage sensor domains, although the mechanisms underlying this form of inactivation may be heterogeneous and are often unclear. Here, we report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K+ channel that lacks a canonical voltage sensor and may thus provide a reduced system to inform on mechanism. In single-channel recordings, we observe that Po decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca2+ or Ba2+, suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K+] (47 mV per 10-fold increase in [K+]), suggesting that K+ binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K+ ≈ Rb+ > Cs+ > Na+ > Li+ ≈ NMG+. Selectivity of the stabilizing site is weaker than that of sites that determine permeability of these ions, suggesting that the site may lie toward the external end of the MthK selectivity filter. We could describe MthK gating over a wide range of positive voltages and external [K+] using kinetic schemes in which the open-conductive state is stabilized by K+ binding to a site that is not deep within the electric field, with the voltage dependence of inactivation arising from both voltage-dependent K+ dissociation and transitions between nonconducting (inactivated) states. These results provide a quantitative working hypothesis for voltage-dependent, K+-sensitive inactivation gating, a property that may be common to other K+ channels

Pharmacology and Surface Electrostatics of the K Channel Outer Pore Vestibule

In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block. Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology. The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge, with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged vestibule with pharmacological properties approaching those of other types of K channels

Predominant Functional Expression of Kv1.3 by Activated Microglia of the Hippocampus after Status epilepticus

BACKGROUND:Growing evidence indicates that the functional state of microglial cells differs according to the pathological conditions that trigger their activation. In particular, activated microglial cells can express sets of Kv subunits which sustain delayed rectifying potassium currents (Kdr) and modulate differently microglia proliferation and ability to release mediators. We recently reported that hippocampal microglia is in a particular activation state after a status epilepticus (SE) and the present study aimed at identifying which of the Kv channels are functionally expressed by microglia in this model. METHODOLOGY/PRINCIPAL FINDINGS:SE was induced by systemic injection of kainate in CX3CR1(eGFP/+) mice and whole cell recordings of fluorescent microglia were performed in acute hippocampal slices prepared 48 h after SE. Microglia expressed Kdr currents which were characterized by a potential of half-maximal activation near -25 mV, prominent steady-state and cumulative inactivations. Kdr currents were almost abolished by the broad spectrum antagonist 4-Aminopyridine (1 mM). In contrast, tetraethylammonium (TEA) at a concentration of 1 mM, known to block Kv3.1, Kv1.1 and 1.2 subunits, only weakly reduced Kdr currents. However, at a concentration of 5 mM which should also affect Kv1.3 and 1.6, TEA inhibited about 30% of the Kdr conductance. Alpha-dendrotoxin, which selectively inhibits Kv1.1, 1.2 and 1.6, reduced only weakly Kdr currents, indicating that channels formed by homomeric assemblies of these subunits are not important contributors of Kdr currents. Finally, agitoxin-2 and margatoxin strongly inhibited the current. CONCLUSIONS/SIGNIFICANCE:These results indicate that Kv1.3 containing channels predominantly determined Kdr currents in activated microglia after SE

Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization

Although action potentials propagate along axons in an all-­or-­none manner, subthreshold membrane potential fluctuations at the soma affect neurotransmitter release from synaptic boutons. An important mechanism underlying analog-­digital modulation is depolarization-­mediated inactivation of presynaptic Kv1-­family potassium channels, leading to action potential broadening and increased calcium influx. Previous studies have relied heavily on recordings from blebs formed after axon transection, which may exaggerate the passive propagation of somatic depolarization. We recorded instead from small boutons supplied by intact axons identified with scanning ion conductance microscopy in primary hippocampal cultures, and asked how distinct potassium channels interact in determining the basal spike width and its modulation by subthreshold somatic depolarization. Pharmacological or genetic deletion of Kv1.1 broadened presynaptic spikes without preventing further prolongation by brief depolarizing somatic prepulses. A heterozygous mouse model of Episodic Ataxia type 1 harboring a dominant Kv1.1 mutation had a similar broadening effect on basal spike shape as deletion of Kv1.1;; however, spike modulation by somatic prepulses was abolished. These results argue that the Kv1.1 subunit is not necessary for subthreshold modulation of spike width. However, a disease-­associated mutant subunit prevents the interplay of analog and digital transmission, possibly by disrupting the normal stoichiometry of presynaptic potassium channels

Potassium Channel and NKCC Cotransporter Involvement in Ocular Refractive Control Mechanisms

Myopia affects well over 30% of adult humans globally. However, the underlying physiological mechanism is little understood. This study tested the hypothesis that ocular growth and refractive compensation to optical defocus can be controlled by manipulation of potassium and chloride ion-driven transretinal fluid movements to the choroid. Chicks were raised with +/−10D or zero power optical defocus rendering the focal plane of the eye in front of, behind, or at the level of the retinal photoreceptors respectively. Intravitreal injections of barium chloride, a non-specific inhibitor of potassium channels in the retina and RPE or bumetanide, a selective inhibitor of the sodium-potassium-chloride cotransporter were made, targeting fluid control mechanisms. Comparison of refractive compensation to 5mM Ba2+ and 10−5 M bumetanide compared with control saline injected eyes shows significant change for both positive and negative lens defocus for Ba2+ but significant change only for negative lens defocus with bumetanide ; ; ; ; ; ). Vitreous chamber depths showed a main effect for drug conditions with less depth change in response to defocus shown for Ba2+ relative to Saline, while bumetanide injected eyes showed a trend to increased depth without a significant interaction with applied defocus. The results indicate that both K channels and the NKCC cotransporter play a role in refractive compensation with NKCC blockade showing far more specificity for negative, compared with positive, lens defocus. Probable sites of action relevant to refractive control include the apical retinal pigment epithelium membrane and the photoreceptor/ON bipolar synapse. The similarities between the biometric effects of NKCC inhibition and biometric reports of the blockade of the retinal ON response, suggest a possible common mechanism. The selective inhibition of refractive compensation to negative lens in chick by loop diuretics such as bumetanide suggests that these drugs may be effective in the therapeutic management of human myopia