Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S)

Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO) or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity. Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions.Methods: Single channel recordings of GH3, GH4 and GH4 STREX cells were used to analyze channel open probability, amplitude and open dwell times. H2S was measured with ananion selective electrode. Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate and evaporation of H2S into account. The results indicate that from a concentration of 300 µM NaHS, only11-13%, i.e. 34-41 µM is effective as H2S in solution. GH3, GH4 and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid.Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S

Ion Channels at the Nucleus: Electrophysiology Meets the Genome

The nuclear envelope is increasingly viewed from an electrophysiological perspective by researchers interested in signal transduction pathways that influence gene transcription and other processes in the nucleus. Here, we describe evidence for ion channels and transporters in the nuclear membranes and for possible ion gating by the nuclear pores. We argue that a systems-level understanding of cellular regulation is likely to require the assimilation of nuclear electrophysiology into molecular and biochemical signaling pathways

BK Channels – Focus on Polyamines, Ethanol/Acetaldehyde and Hydrogen Sulfide (H2S)

(VLID)386021

S100 Calcium Binding Proteins and Ion Channels

S100 Ca2+-binding proteins have been associated with a multitude of intracellular Ca2+-dependent functions including regulation of the cell cycle, cell differentiation, cell motility and apoptosis, modulation of membrane–cytoskeletal interactions, transduction of intracellular Ca2+ signals, and in mediating learning and memory. S100 proteins are fine tuned to read the intracellular free Ca2+ concentration and affect protein phosphorylation, which makes them candidates to modulate certain ion channels and neuronal electrical behavior. Certain S100s are secreted from cells and are found in extracellular fluids where they exert unique extracellular functions. In addition to their neurotrophic activity, some S100 proteins modulate neuronal electrical discharge activity and appear to act directly on ion channels. The first reports regarding these effects suggested S100-mediated alterations in Ca2+ fluxes, K+ currents, and neuronal discharge activity. Recent reports revealed direct and indirect interactions with Ca2+, K+, Cl−, and ligand activated channels. This review focuses on studies of the physical and functional interactions of S100 proteins and ion channels

Mechanisms of Two Modulatory Actions of the Channel-binding Protein Slob on the Drosophila Slowpoke Calcium-dependent Potassium Channel

Slob57 is an ion channel auxiliary protein that binds to and modulates the Drosophila Slowpoke calcium-dependent potassium channel (dSlo). We reported recently that residues 1–39 of Slob57 comprise the key domain that both causes dSlo inactivation and shifts its voltage dependence of activation to more depolarized voltages. In the present study we show that removal of residues 2–6 from Slob57 abolishes the inactivation, but the ability of Slob57 to rightward shift the voltage dependence of activation of dSlo remains. A synthetic peptide corresponding in sequence to residues 1–6 of Slob57 blocks dSlo in a voltage- and dose-dependent manner. Two Phe residues and at least one Lys residue in this peptide are required for the blocking action. These data indicate that the amino terminus of Slob57 directly blocks dSlo, thereby leading to channel inactivation. Further truncation to residue Arg16 eliminates the modulation of voltage dependence of activation. Thus these two modulatory actions of Slob57 are independent. Mutation within the calcium bowl of dSlo greatly reduces its calcium sensitivity (Bian, S., I. Favre, and E. Moczydlowski. 2001. Proc. Natl. Acad. Sci. USA. 98:4776–4781). We found that Slob57 still causes inactivation of this mutant channel, but does not shift its voltage dependence of activation. This result confirms further the independence of the inactivation and the voltage shift produced by Slob57. It also suggests that the voltage shift requires high affinity Ca2+ binding to an intact calcium bowl. Furthermore, Slob57 inhibits the shift in the voltage dependence of activation of dSlo evoked by Ca2+, and this inhibition by Slob57 is greater at higher free Ca2+ concentrations. These results implicate distinct calcium-dependent and -independent mechanisms in the modulation of dSlo by Slob

Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions

Emerging role of the calcium-activated, small conductance, SK3 K ⁺ channel in distal tubule function: Regulation by TRPV4

The Ca2+-activated, maxi-K (BK) K+ channel, with low Ca2+-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K+ secretion. In the present study we demonstrate that the Ca2+-activated, SK3 (KCa2.3) K + channel, with high Ca2+-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K+ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca2+- dependent K+ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca2+-permeable TRPV4 channel, thereby inducing Ca2+ influx and elevating intracellular Ca2+ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+- affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion. © 2014 Berrout et al

Is Consciousness Dissectible? Acute Slice Electrophysiology and a Bayesian Interpretation of Neural Correlates of Consciousness

The acute brain slicing method has become one of the foundations of modern neuroscience research. It is a laboratory technique in electrophysiology, which allows the study of electrical properties directly on a freshly prepared slice of animal brain tissue. During recording and/or stimulation, the acutely isolated brain slice is artificially kept “alive†up to many hours after the animals’ death. During an acute brain slice preparation, cortical and subcortical areas, which are suggested to correlate with conscious experience in humans, such as the claustrum and the thalamus, are dissected. In this paper, we investigate whether scientific statements can be made regarding the likelihood that some neural activities on the brain slice still support consciousness or degrees thereof.We exemplarily demonstrate how acute slices are produced and provide own electrophysiological data combined with a short literature review. Subsequently, we introduce the concept of Neural Correlates of Consciousness (NCC) and apply conditional probabilities inferred from Bayes´ theorem, in order to draw from it an informed hypothesis on the likelihood that specific neural activities that sustain on the slice still correlate with some form of conscious experience. We propose that the probability that there is something that is it like to be, even on the acutely isolated brain slice, is similar to the likelihood that certain mental states correlate with certain brain activities in a healthy human subject, depending on the robustness of the underlying NCC