Intrinsic Electrostatic Potential in the BK Channel Pore: Role in Determining Single Channel Conductance and Block

The internal vestibule of large-conductance Ca2+ voltage-activated K+ (BK) channels contains a ring of eight negative charges not present in K+ channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017–9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K+ was 18% lower than the control. Current–voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K+ (30 mM) and vanished at high K+ (1 M K+). We determine the electrostatic potential change, Δφ, caused by the charge neutralization using TEA+ block for the extracellular charges and Ba2+ for intracellular charges. We measured 13 ± 2 mV for Δφ at the TEA+ site when turning off the extracellular charges, and 17 ± 2 mV for the Δφ at the Ba2+ site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Δφ using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329

Wheat Germ Agglutinin Conjugated Fluorescent pH Sensors for Visualizing Proton Fluxes [preprint]

Small molecule fluorescent wheat germ agglutinin (WGA) conjugates are routinely used to demarcate mammalian plasma membranes because they bind to the cell’s glycocalyx. Here we describe the derivatization of WGA with a pH sensitive rhodamine fluorophore (pHRho: pKa = 7) to detect proton channel fluxes and extracellular proton accumulation and depletion from primary cells. We found that WGA-pHRho labeling was uniform, did not appreciably alter the voltage-gating of glycosylated ion channels, and the extracellular changes in pH directly correlated with proton channel activity. Using single plane illumination techniques, WGA-pHRho was used to detect spatiotemporal differences in proton accumulation and depletion over the extracellular surface of cardiomyocytes, astrocytes, and neurons. Because WGA can be derivatized with any small molecule fluorescent ion sensor, WGA conjugates should prove useful to visualize most electrogenic and non-electrogenic events on the extracellular side of the plasma membrane

Near-Membrane Dynamics and Capture of TRPM8 Channels within Transient Confinement Domains

The cold and menthol receptor, TRPM8, is a non-selective cation channel expressed in a subset of peripheral neurons that is responsible for neuronal detection of environmental cold stimuli. It was previously shown that members of the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process. Single particle tracking (SPT) is a useful tool for probing the organization and dynamics of protein constituents in the plasma membrane.We used SPT to study the receptor dynamics and describe membrane/near-membrane behavior of particles containing TRPM8-EGFP in transfected HEK-293T and F-11 cells. Cells were imaged using total internal reflection fluorescence (TIRF) microscopy and the 2D and 3D trajectories of TRPM8 molecules were calculated by analyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel, most TRPM8 particles move in network covering the PM, periodically lingering for 2–8 s in confined microdomains of about 800 nm radius. Removing cholesterol with methyl-beta-cyclodextrin (MβCD) stabilizes TRPM8 motion in the PM and is correlated with larger TRPM8 current amplitude that results from an increase in the number of available channels without a change in open probability.These results reveal a novel mechanism for regulating TRPM8 channel activity, and suggest that PM dynamics may play an important role in controlling electrical activity in cold-sensitive neurons

The Integration of Electrical Signals Originating in the Root of Vascular Plants

Plants have developed different signaling systems allowing for the integration of environmental cues to coordinate molecular processes associated to both early development and the physiology of the adult plant. Research on systemic signaling in plants has traditionally focused on the role of phytohormones as long-distance signaling molecules, and more recently the importance of peptides and miRNAs in building up this communication process has also been described. However, it is well-known that plants have the ability to generate different types of long-range electrical signals in response to different stimuli such as light, temperature variations, wounding, salt stress, or gravitropic stimulation. Presently, it is unclear whether short or long-distance electrical communication in plants is linked to nutrient uptake. This review deals with aspects of sensory input in plant roots and the propagation of discrete signals to the plant body. We discuss the physiological role of electrical signaling in nutrient uptake and how nutrient variations may become an electrical signal propagating along the plant

Structurally conserved C-RFa revealed prolactin releasing activity in vitro and gene expression changes in pituitary of seasonally acclimatized carp.

Here we show the cloning and characterization of a novel homolog of prepro C-RFa cDNA from Cyprinus carpio. The deduced preprohormone precursor of 115 amino acids leads to a mature bioactive peptide of 20 amino acids with identical sequence to other teleost C-RFa. Modeling of the mature C-RFa peptide highlighted significant similarity to homologous human PrRP20, specifically the conserved amphipathic system defined by the C-terminal alpha-helix. Clearly, the synthetic C-RFa peptide stimulated prolactin release from primary cultured fish pituitary cells. For the first time, significant variation was shown in C-RFa mRNA and protein levels in the hypothalamus and pituitary between summer- and winter-acclimatized carp. Furthermore, C-RFa protein distribution in carp central nervous tissue was visualized by immunodetection in fibers and cells in hypothalamus, olfactory tract, cerebellum and pituitary stalk. In conclusion, we demonstrated the structure conservation of C-RFa in teleosts and mammals and immunopositive cells and fibers for C-RFa in brain areas. Finally, the increase of C-RFa expression suggests the participation of this hypothalamic factor in the mechanism of modulation in PRL expression in carp

A cell-free assay to determine the stoichiometry of plasma membrane proteins

Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice