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Kv4 channels are thought to lack a C-type inactivation mechanism (collapse of the external pore) and to inactivate as a result of a concerted action of cytoplasmic regions of the channel. To investigate whether Kv4 channels have outer pore conformational changes during the inactivation process, the inactivation properties of Kv4.3 were characterized in 0 mM and in 2 mM external K+ in whole-cell voltage-clamp experiments. Removal of external K+ increased the inactivation rates and favored cumulative inactivation by repetitive stimulation. The reduction in current amplitude during repetitive stimulation and the faster inactivation rates in 0 mM external K+ were not due to changes in the voltage dependence of channel opening or to internal K+ depletion. The extent of the collapse of the K+ conductance upon removal of external K+ was more pronounced in NMG+-than in Na+-containing solutions. The reduction in the current amplitude during cumulative inactivation by repetitive stimulation is not associated with kinetic changes, suggesting that it is due to a diminished number of functional channels with unchanged gating properties. These observations meet the criteria for a typical C-type inactivation, as removal of external K+ destabilizes the conducting state, leading to the collapse of the pore. A tentative model is presented, in which K+ bound to high-affinity K+-binding sites in the selectivity filter destabilizes an outer neighboring K+ modulatory site that is saturated at ~2 mM external K+. We conclude that Kv4 channels have a C-type inactivation mechanism and that previously reported alterations in the inactivation rates after N- and C- termini mutagenesis may arise from secondary changes in the electrostatic interactions between K+-binding sites in the selectivity filter and the neighboring K+-modulatory site, that would result in changes in its K+ occupancy. [ABSTRACT FROM AUTHOR]

Voltage-dependent and calcium-activated K⁺ (MaxiK, BK) channels are widely expressed in many tissues and organs where they play various physiological roles. Here we report discovery of a functional trafficking signal in MaxiK channel accessory β4 subunit that could regulate activity of MaxiK α subunit (hSlo) on the plasma membrane. We demonstrate that β4 is mostly retained within the cell and removal or mutation of β4 trafficking signal significantly enhances its surface expression in HEK293T expression system. In hippocampal slices and cultured neurons we also observed significant β4 expressions within the neurons. Finally, we show that unlike SV1 and β1 subunits, β4 shows no dominant-negative effect on MaxiK channel α subunit. Taken together, we propose β4 subunit of MaxiK channel is mostly retained within the cells without interfering with other subunits. Removal of β4 retention signal increases its surface expression that may lead to reduction of the MaxiK channel activity and neuronal excitability., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Large conductance voltage and calcium-activated K(+) channels play critical roles in neuronal excitability and vascular tone. Previously, we showed that coexpression of the transmembrane beta2 subunit, KCNMB2, with the human pore-forming alpha subunit of the large conductance voltage and Ca(2+)-activated K(+) channel (hSlo) yields inactivating currents similar to those observed in hippocampal neurons [Hicks GA, Marrion NV (1998) Ca(2+)-dependent inactivation of large conductance Ca(2+)-activated K(+) (BK) channels in rat hippocampal neurones produced by pore block from an associated particle. J Physiol (Lond) 508 (Pt 3):721-734; Wallner M, Meera P, Toro L (1999b) Molecular basis of fast inactivation in voltage and Ca(2+)-activated K(+) channels: A transmembrane beta-subunit homolog. Proc Natl Acad Sci U S A 96:4137-4142]. Herein, we report that coexpression of beta2 subunit with hSlo can also modulate hSlo surface expression levels in HEK293T cells. We found that, when expressed alone, beta2 subunit appears to reach the plasma membrane but also displays a distinct intracellular punctuated pattern that resembles endosomal compartments. beta2 Subunit coexpression with hSlo causes two biological effects: i) a shift of hSlo's intracellular expression pattern from a relatively diffuse to a distinct punctated cytoplasmic distribution overlapping beta2 expression; and ii) a decrease of hSlo surface expression that surpassed an observed small decrease in total hSlo expression levels. beta2 Site-directed mutagenesis studies revealed two putative endocytic signals at the C-terminus of beta2 that can control expression levels of hSlo. In contrast, a beta2 N-terminal consensus endocytic signal had no effect on hSlo expression levels. Thus, beta2 subunit not only can influence hSlo currents but also has the ability to limit hSlo surface expression levels via an endocytic mechanism. This new mode of beta2 modulation of hSlo may depend on particular coregulatory mechanisms in different cell types.

Voltage-dependent and calcium-activated K(+) (MaxiK, BK) channels are ubiquitously expressed and have various physiological roles including regulation of neurotransmitter release and smooth muscle tone. Coexpression of the pore-forming alpha (hSlo) subunit of MaxiK channels with a regulatory beta1 subunit (KCNMB1) produces noninactivating currents that are distinguished by high voltage/Ca(2+) sensitivities and altered pharmacology [McManus OB, Helms LM, Pallanck L, Ganetzky B, Swanson R, Leonard RJ (1995) Functional role of the beta subunit of high conductance calcium-activated potassium channels. Neuron 14:645-650; Wallner M, Meera P, Ottolia M, Kaczorowski G, Latorre R, Garcia ML, Stefani E, Toro L (1995) Characterization of and modulation by a beta-subunit of a human maxi K(Ca) channel cloned from myometrium. Receptors Channels 3:185-199]. We now show that beta1 can regulate hSlo traffic as well, resulting in decreased hSlo surface expression. beta1 subunit expressed alone is able to reach the plasma membrane; in addition, it exhibits a distinct intracellular punctated pattern that colocalizes with an endosomal marker. Coexpressing beta1 subunit with hSlo, switches hSlo's rather diffuse intracellular expression to a punctate cytoplasmic localization that overlaps beta1 expression. Furthermore, coexpressed beta1 subunit reduces steady-state hSlo surface expression. Site-directed mutagenesis underscores a role of a putative endocytic signal at the beta1 C-terminus in the control of hSlo surface expression. We propose that aside from its well-established role as regulator of hSlo electrical activity, beta1 can regulate hSlo expression levels by means of an endocytic mechanism. This highlights a new beta1 subunit feature that regulates hSlo channels by a trafficking mechanism.

Though there is some evidence to the contrary, dogma claims that primary sensory neurons in the dorsal root ganglion do not interact, that the ganglion serves as a through-station in which no signal processing occurs. Here we use patch clamp and immunocytochemistry to show that sensory neurons in primary culture can form chemical synapses on each other. The resulting neurotransmitter release is calcium dependent and uses synaptotagmin-containing vesicles. On many cells studied, the postsynaptic receptor for the neurotransmitter is a P2X receptor, an ion channel activated by extracellular ATP. This shows that sensory neurons have the machinery to form purinergic synapses on each other and that they do so when placed in short-term tissue culture.

We identified a novel MaxiK alpha subunit splice variant (SV1) from rat myometrium that is also present in brain. SV1 has a 33-amino acid insert in the S1 transmembrane domain that does not alter S1 overall hydrophobicity, but makes the S0-S1 linker longer. SV1 was transfected in HEK293T cells and studied using immunocytochemistry and electrophysiology. In non-permeabilized cells, N-terminal c-Myc- or C-terminal green fluorescent protein-tagged SV1 displayed no surface labeling or currents. The lack of SV1 functional expression was due to endoplasmic reticulum (ER) retention as determined by colabeling experiments with a specific ER marker. To explore the functional role of SV1, we coexpressed SV1 with the alpha (human SLO) and beta1 (KCNMB1) subunits of the MaxiK channel. Coexpression of SV1 inhibited surface expression of alpha and beta1 subunits approximately 80% by trapping them in the ER. This inhibition seems to be specific for MaxiK channel subunits since SV1 was unable to prevent surface expression of the Kv4.3 channel or to interact with green fluorescent protein. These results indicate a dominant-negative role of SV1 in MaxiK channel expression. Moreover, they reveal down-regulation by splice variants as a new mechanism that may contribute to the diverse levels of MaxiK channel expression in non-excitable and excitable cells.

The hippocampus receives cholinergic afferents and expresses neuronal nicotinic acetylcholine receptors. In particular, the alpha7 and beta2 nicotinic subunits are highly expressed in the hippocampus. There has been controversy about the location, distribution and roles of neuronal nicotinic acetylcholine receptors [Role L. W. and Berg D. K. (1996) Neuron 16, 1077-1085; Wonnacott S. (1997) Trends Neurosci. 20, 92-98]. Using immunocytochemistry and patch-clamp techniques, we examined the density and distribution of nicotinic receptors on rat hippocampal neurons in primary tissue culture. The density and distribution of alpha7 subunits change with days in culture. Before 10 days in culture, alpha7 expression, monitored immunocytochemically, is low and nicotinic currents are small or absent. In older cultures, about two-thirds of the neurons express nicotinic currents, and alpha7 appears in small patches on the soma and out along the neuronal processes. These patches of alpha7 subunits on the surface of the neuronal processes often co-localize with the presynaptic marker, synaptotagmin. The other most common nicotinic subunit, beta2, stays confined mainly to the soma and proximal processes, and beta2 is distributed more uniformly and is not specifically localized at presynaptic areas. The two subunits, alpha7 and beta2, have different expression patterns on the surface of the cultured hippocampal neurons. Taken together with previous physiological studies, the results indicate that alpha7 subunits can be found at presynaptic terminals, and at these locations, these calcium-permeable channels may influence transmitter release.

Genetic manipulation offers great potential for studying the molecular and cellular processes which control or regulate the complex developmental properties of neurons. Gene transfer into neurons, however, is notoriously difficult. In this study we have used a replication-defective adenovirus (Adv/RSV beta gal), expressing beta-galactosidase (beta-gal) as a reporter gene, to infect dissociated cultures of rat hippocampal neurons and hippocampal slice cultures. Because future studies will require either long-term (e.g., developmental) or short-term (e.g., electrophysiological) expression of recombinant genes in neuronal cultures, we have optimized infection conditions for each situation. The Adv/RSV beta gal construct infects neurons and glial cells equally well, with no apparent alterations in cellular morphology. In slice cultures, the same efficiency and temporal control of beta-gal expression following Adv/RSV beta gal infection was achieved. Focal application of the adenoviruses, by microinjection, permitted infection of discrete subregions within the hippocampal explants. Whole cell recordings of dissociated hippocampal neurons and field recordings from the explant cultures, infected with Adv/RSV beta gal at low multiplicities of infection, indicated no significant alteration in the electrophysiological profiles of neurons in these cultures. The results demonstrate the utility of adenoviruses as gene transfer vectors for primary cultures of neurons. Adenovirus-mediated gene transfer into slice cultures also provides an opportunity to study development or plasticity in an environment where the circuitry and cytoarchitecture of the tissue are preserved and the areas of genetic manipulation can be spatially isolated.

The open-channel structure of the N-methyl-D-aspartate (NMDA) receptor was investigated to explain apparently conflicting interpretations about ionic interactions within the pore. Patch-clamp techniques were applied to tissue-cultured rat hippocampal neurons from the CA1 region. A wide range of ammonium derivatives was studied to learn about the structure of the pore from permeability and open-channel blocking characteristics. We conclude that the pore is asymmetric, having high-affinity binding for organic cations from the outside and having a larger external entrance. The minimum cross-sectional area of the pore is rectangular (approximately 0.45 x 0.57 nm) and is the single-occupancy binding site(s) for small permeant cations. The narrow region extending from this minimum cross section is short, and its shape underlies the voltage dependencies of blocking cations. While occupying the blocking site, some open-channel blockers can interact with permeant cations at their binding site in the minimum cross section. A structurally based hypothesis is presented, explaining that the electrostatic interactions between the blocking site and permeant-ion site produce a high voltage dependence for blockade by some cations.