The exocyst complex is required for the trafficking and delivery of KCa3.1 to the basolateral membrane of polarized epithelia.

Control of the movement of ions and water across epithelia is essential for homeostasis. Changing the number or activity of ion channels at the plasma membrane is a significant regulator of epithelial transport. In polarized epithelia, the intermediate-conductance calcium-activated potassium channel, KCa3.1 is delivered to the basolateral membrane where it generates and maintains the electrochemical gradients required for epithelial transport. The mechanisms that control the delivery of KCa3.1 to the basolateral membrane are still emerging. Herein, we investigated the role of the highly conserved tethering complex exocyst. In epithelia, exocyst is involved in the tethering of post-Golgi secretory vesicles with the basolateral membrane, which is required before membrane fusion. In our Fisher rat thyroid cell line that stably expresses KCa3.1, siRNA knockdown of either of the exocyst subunits Sec3, Sec6, or Sec8 significantly decreased KCa3.1-specific current. In addition, knockdown of exocyst complex subunits significantly reduced the basolateral membrane protein level of KCa3.1. Finally, co-immunoprecipitation experiments suggest associations between Sec6 and KCa3.1, but not between Sec8 and KCa3.1. Collectively, based on these data and our previous studies, we suggest that components of exocyst complex are crucially important in the tethering of KCa3.1 to the basolateral membrane. After which, Soluble N-ethylmaleimide-sensitive factor (SNF) Attachment Receptors (SNARE) proteins aid in the insertion of KCa3.1-containing vesicles into the basolateral membrane of polarized epithelia.NEW & NOTEWORTHY Our Ussing chamber and immunoblot experiments demonstrate that when subunits of the exocyst complex were transiently knocked down, this significantly reduced the basolateral population and functional expression of KCa3.1. These data suggest, combined with our protein association experiments, that the exocyst complex regulates the tethering of KCa3.1-containing vesicles to the basolateral membrane prior to the SNARE-dependent insertion of channels into the basolateral membrane of epithelial cells.

KCa3.1 potentiation stimulates Cl- secretion in F508del and G551D CFTR-corrected primary human bronchial epithelial cells.

We previously identified potentiators of KCa3.1 (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; DCEBIO) that stimulate Cl- secretion across human bronchial epithelial cells (HBEs) expressing wild-type (WT) cystic fibrosis transmembrane conductance regulator (CFTR). However, these compounds failed to stimulate Cl- secretion in F508del CFTR HBEs. Drug discovery efforts identified CFTR potentiators (VX-770) and correctors (VX-445, VX-661) for cystic fibrosis (CF) disease-causing mutations, including F508del and G551D. Herein, we evaluated the effect of KCa3.1 potentiation on Cl- equivalent current (ICl) across primary HBEs expressing WT, F508del, and G551D CFTR. Transepithelial impedance analysis was used to obtain estimates of apical (Ra) and basolateral membrane (BLM; Rb) resistances. In WT CFTR HBEs, DCEBIO stimulated ICl, which was increased by forskolin. Similarly, forskolin stimulated ICl, and this was increased by DCEBIO. The KCa3.1 blocker, TRAM-34 inhibited ICl. DCEBIO decreased Rb, whereas TRAM-34 increased Rb, consistent with BLM localization of KCa3.1. Following correction of F508del CFTR with VX-445 + VX-661, DCEBIO failed to stimulate ICl, although the subsequent addition of forskolin + VX-770 increased ICl. Importantly, following stimulation of ICl with forskolin + VX-770, DCEBIO induced a further significant increase in ICl. As above, DCEBIO reduced Rb, whereas TRAM-34 increased Rb, consistent with BLM localized KCa3.1. Finally, we assessed KCa3.1 potentiation on ICl in G551D/F508del CFTR HBEs in the absence or presence of VX-445 + VX-661. In both cases, DCEBIO failed to stimulate ICl. However, following stimulation with forskolin + VX-770, DCEBIO nearly doubled ICl. Our results demonstrate that following correction/potentiation of F508del and G551D CFTR, potentiation of KCa3.1 increases the Cl- secretory response, suggesting this class of compounds may represent a novel means of further increasing Cl- secretion across CF airway.

Histone deacetylase inhibitors (HDACi) increase expression of KCa2.3 (SK3) in primary microvascular endothelial cells.

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation.

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator (CFTR) correctors VX-445 and VX-121.

Cystic fibrosis (CF) results from mutations in the CFTR anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The BKCa potassium channel is also critical for maintaining lung ASL volume. Here, we show the CFTR corrector, VX-445 (Elexacaftor), a component of Trikafta, induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121 - a corrector under clinical evaluation. Whole-cell patch-clamp recordings confirmed potentiated channel activity from CFTR correctors on the BKCa α-subunit, and excised patch-clamp recordings demonstrated a significant increase in open probability. In mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced action potential firing frequency in primary hippocampal and cortical neurons. VX-445 effects were observed at low micomolar concentrations (1-10 µM) - within the range reported in plasma and tissues from CF patients. We raise the possibilities that CFTR correctors gain additional clinical benefit by activation of BKCa in the lung, yet may lead to adverse events through BKCa activation, elsewhere.

Basolateral membrane K+ channels in renal epithelial cells.

The major function of epithelial tissues is to maintain proper ion, solute, and water homeostasis. The tubule of the renal nephron has an amazingly simple structure, lined by epithelial cells, yet the segments (i.e., proximal tubule vs. collecting duct) of the nephron have unique transport functions. The functional differences are because epithelial cells are polarized and thus possess different patterns (distributions) of membrane transport proteins in the apical and basolateral membranes of the cell. K(+) channels play critical roles in normal physiology. Over 90 different genes for K(+) channels have been identified in the human genome. Epithelial K(+) channels can be located within either or both the apical and basolateral membranes of the cell. One of the primary functions of basolateral K(+) channels is to recycle K(+) across the basolateral membrane for proper function of the Na(+)-K(+)-ATPase, among other functions. Mutations of these channels can cause significant disease. The focus of this review is to provide an overview of the basolateral K(+) channels of the nephron, providing potential physiological functions and pathophysiology of these channels, where appropriate. We have taken a "K(+) channel gene family" approach in presenting the representative basolateral K(+) channels of the nephron. The basolateral K(+) channels of the renal epithelia are represented by members of the KCNK, KCNJ, KCNQ, KCNE, and SLO gene families.

Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1.

We recently demonstrated that plasma membrane KCa3.1 is rapidly endocytosed and targeted for lysosomal degradation via a Rab7- and ESCRT-dependent pathway. Herein, we assess the role of ubiquitylation in this process. Using a biotin ligase acceptor peptide (BLAP)-tagged KCa3.1, in combination with tandem ubiquitin binding entities (TUBEs), we demonstrate that KCa3.1 is polyubiquitylated following endocytosis. Hypertonic sucrose inhibited KCa3.1 endocytosis and resulted in a significant decrease in channel ubiquitylation. Inhibition of the ubiquitin-activating enzyme (E1) with UBEI-41 resulted in reduced KCa3.1 ubiquitylation and internalization. The general deubiquitylase (DUB) inhibitor, PR-619 attenuated KCa3.1 degradation, indicative of deubiquitylation being required for lysosomal delivery. Using the DUB Chip, a protein microarray containing 35 DUBs, we demonstrate a time-dependent association between KCa3.1 and USP8 following endocytosis, which was confirmed by coimmunoprecipitation. Further, overexpression of wild-type USP8 accelerates channel deubiquitylation, while either a catalytically inactive mutant USP8 or siRNA-mediated knockdown of USP8 enhanced accumulation of ubiquitylated KCa3.1, thereby inhibiting channel degradation. In summary, by combining BLAP-tagged KCa3.1 with TUBEs and DUB Chip methodologies, we demonstrate that polyubiquitylation mediates the targeting of membrane KCa3.1 to the lysosomes and also that USP8 regulates the rate of KCa3.1 degradation by deubiquitylating KCa3.1 prior to lysosomal delivery.

Role of SNARE Proteins in the Insertion of KCa3.1 in the Plasma Membrane of a Polarized Epithelium.

Targeting proteins to a specific membrane is crucial for proper epithelial cell function. KCa3.1, a calcium-activated, intermediate-conductance potassium channel, is targeted to the basolateral membrane (BLM) in epithelial cells. Surprisingly, the mechanism of KCa3.1 membrane targeting is poorly understood. We previously reported that targeting of KCa3.1 to the BLM of epithelial cells is Myosin-Vc-, Rab1-and Rab8-dependent. Here, we examine the role of the SNARE proteins VAMP3, SNAP-23 and syntaxin 4 (STX-4) in the targeting of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We carried out immunoblot, siRNA and Ussing chamber experiments on FRT cells, stably expressing KCa3.1-BLAP/Bir-A-KDEL, grown as high-resistance monolayers. siRNA-mediated knockdown of VAMP3 reduced BLM expression of KCa3.1 by 57 ± 5% (p ≤ 0.05, n = 5). Measurements of BLM-localized KCa3.1 currents, in Ussing chambers, demonstrated knockdown of VAMP3 reduced KCa3.1 current by 70 ± 4% (p ≤ 0.05, n = 5). Similarly, siRNA knockdown of SNAP-23 reduced the expression of KCa3.1 at the BLM by 56 ± 7% (p ≤ 0.01, n = 6) and reduced KCa3.1 current by 80 ± 11% (p ≤ 0.05, n = 6). Also, knockdown of STX-4 lowered the BLM expression of KCa3.1 by 54 ± 6% (p ≤ 0.05, n = 5) and reduced KCa3.1 current by 78 ± 11% (p ≤ 0.05, n = 5). Finally, co-immunoprecipitation experiments demonstrated associations between KCa3.1, VAMP3, SNAP-23 and STX-4. These data indicate that VAMP3, SNAP-23 and STX-4 are critical for the targeting KCa3.1 to BLM of polarized epithelial cells.

Calcium-activated K+ channels increase cell proliferation independent of K+ conductance.

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.

Recycling of the Ca2+-activated K+ channel, KCa2.3, is dependent upon RME-1, Rab35/EPI64C, and an N-terminal domain.

Regulation of the number of Ca(2+)-activated K(+) channels at the endothelial cell surface contributes to control of the endothelium-derived hyperpolarizing factor response, although this process is poorly understood. To address the fate of plasma membrane-localized KCa2.3, we utilized an extracellular epitope-tagged channel in combination with fluorescence and biotinylation techniques in both human embryonic kidney cells and the human microvascular endothelial cell line, HMEC-1. KCa2.3 was internalized from the plasma membrane and degraded with a time constant of 18 h. Cell surface biotinylation demonstrated that KCa2.3 was rapidly endocytosed and recycled back to the plasma membrane. Consistent with recycling, expression of a dominant negative (DN) RME-1 or Rab35 as well as wild type EPI64C, the Rab35 GTPase-activating protein, resulted in accumulation of KCa2.3 in an intracellular compartment. Expression of DN RME-1, DN Rab35, or wild type EPI64C resulted in a decrease in steady-state plasma membrane expression. Knockdown of EPI64C increased cell surface expression of KCa2.3. Furthermore, the effect of EPI64C was dependent upon its GTPase-activating proteins activity. Co-immunoprecipitation studies confirmed an association between KCa2.3 and both Rab35 and RME-1. In contrast to KCa2.3, KCa3.1 was rapidly endocytosed and degraded in an RME-1 and Rab35-independent manner. A series of N-terminal deletions identified a 12-amino acid region, Gly(206)-Pro(217), as being required for the rapid recycling of KCa2.3. Deletion of Gly(206)-Pro(217) had no effect on the association of KCa2.3 with Rab35 but significantly decreased the association with RME-1. These represent the first studies elucidating the mechanisms by which KCa2.3 is maintained at the plasma membrane.

ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes.

The number of intermediate-conductance, Ca(2+)-activated K(+) channels (KCa3.1) present at the plasma membrane is deterministic in any physiological response. However, the mechanisms by which KCa3.1 channels are removed from the plasma membrane and targeted for degradation are poorly understood. Recently, we demonstrated that KCa3.1 is rapidly internalized from the plasma membrane, having a short half-life in both human embryonic kidney cells (HEK293) and human microvascular endothelial cells (HMEC-1). In this study, we investigate the molecular mechanisms controlling the degradation of KCa3.1 heterologously expressed in HEK and HMEC-1 cells. Using immunofluorescence and electron microscopy, as well as quantitative biochemical analysis, we demonstrate that membrane KCa3.1 is targeted to the lysosomes for degradation. Furthermore, we demonstrate that either overexpressing a dominant negative Rab7 or short interfering RNA-mediated knockdown of Rab7 results in a significant inhibition of channel degradation rate. Coimmunoprecipitation confirmed a close association between Rab7 and KCa3.1. On the basis of these findings, we assessed the role of the ESCRT machinery in the degradation of heterologously expressed KCa3.1, including TSG101 [endosomal sorting complex required for transport (ESCRT)-I] and CHMP4 (ESCRT-III) as well as VPS4, a protein involved in the disassembly of the ESCRT machinery. We demonstrate that TSG101 is closely associated with KCa3.1 via coimmunoprecipitation and that a dominant negative TSG101 inhibits KCa3.1 degradation. In addition, both dominant negative CHMP4 and VPS4 significantly decrease the rate of membrane KCa3.1 degradation, compared with wild-type controls. These results are the first to demonstrate that plasma membrane-associated KCa3.1 is targeted for lysosomal degradation via a Rab7 and ESCRT-dependent pathway.

Plasma membrane insertion of KCa2.3 (SK3) is dependent upon the SNARE proteins, syntaxin-4 and SNAP23.

We previously demonstrated endocytosis of KCa2.3 is caveolin-1-, dynamin II- and Rab5-dependent. KCa2.3 then enters Rab35/EPI64C- and RME-1-containing recycling endosomes and is returned to the plasma membrane (PM). Herein, we report on the mechanism by which KCa2.3 is inserted into the PM during recycling and following exit from the Golgi. We demonstrate KCa2.3 colocalizes with SNAP-23 and Syntaxin-4 in the PM of HEK and endothelial cells by confocal immunofluorescence microscopy. We further show KCa2.3 can be co-immunoprecipitated with SNAP-23 and Syntaxin-4. Overexpression of either Syntaxin-4 or SNAP-23 increased PM expression of KCa2.3, whereas shRNA-mediated knockdown of these SNARE proteins significantly decreased PM KCa2.3 expression, as assessed by cell surface biotinylation. Whole-cell patch clamp studies confirmed knockdown of SNAP-23 significantly decreased the apamin sensitive, KCa2.3 current. Using standard biotinylation/stripping methods, we demonstrate shRNA mediated knockdown of SNAP-23 inhibits recycling of KCa2.3 following endocytosis, whereas scrambled shRNA had no effect. Finally, using biotin ligase acceptor peptide (BLAP)-tagged KCa2.3, coupled with ER-resident biotin ligase (BirA), channels could be biotinylated in the ER after which we evaluated their rate of insertion into the PM following Golgi exit. We demonstrate knockdown of SNAP-23 significantly slows the rate of Golgi to PM delivery of KCa2.3. The inhibition of both recycling and PM delivery of newly synthesized KCa2.3 channels likely accounts for the decreased PM expression observed following knockdown of these SNARE proteins. In total, our results suggest insertion of KCa2.3 into the PM depends upon the SNARE proteins, Syntaxin-4 and SNAP-23.

Modulation of Retrograde Trafficking of KCa3.1 in a Polarized Epithelium.

In epithelia, the intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane (BLM) where this channel plays numerous roles in absorption and secretion. A growing body of research suggests that the membrane resident population of KCa3.1 may be critical in clinical manifestation of diseases. In this study, we investigated the key molecular components that regulate the degradation of KCa3.1 using a Fisher rat thyroid cell line stably expressing KCa3.1. Using immunoblot, Ussing chamber, and pharmacological approaches, we demonstrated that KCa3.1 is targeted exclusively to the BLM, provided a complete time course of degradation of KCa3.1 and degradation time courses of the channel in the presence of pharmacological inhibitors of ubiquitylation and deubiquitylation to advance our understanding of the retrograde trafficking of KCa3.1. We provide a complete degradation profile of KCa3.1 and that the degradation is via an ubiquitin-dependent pathway. Inhibition of E1 ubiquitin activating enzyme by UBEI-41 crippled the ability of the cells to internalize the channel, shown by the increased BLM surface expression resulting in an increased function of the channel as measured by a DCEBIO sensitive K+ current. Additionally, the involvement of deubiquitylases and degradation by the lysosome were also confirmed by treating the cells with PR-619 or leupeptin/pepstatin, respectively; which significantly decreased the degradation rate of membrane KCa3.1. Additionally, we provided the first evidence that KCa3.1 channels were not deubiquitylated at the BLM. These data further define the retrograde trafficking of KCa3.1, and may provide an avenue for therapeutic approach for treatment of disease.

Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent.

The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the µ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of µ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells.

A small conductance calcium-activated K+ channel in C. elegans, KCNL-2, plays a role in the regulation of the rate of egg-laying.

In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying.

Trafficking of intermediate (KCa3.1) and small (KCa2.x) conductance, Ca(2+)-activated K(+) channels: a novel target for medicinal chemistry efforts?

Ca(2+)-activated K(+) (KCa) channels play a pivotal role in the physiology of a wide variety of tissues and disease states, including vascular endothelia, secretory epithelia, certain cancers, red blood cells (RBC), neurons, and immune cells. Such widespread involvement has generated an intense interest in elucidating the function and regulation of these channels, with the goal of developing pharmacological strategies aimed at selective modulation of KCa channels in various disease states. Herein we give an overview of the molecular and functional properties of these channels and their therapeutic importance. We discuss the achievements made in designing pharmacological tools that control the function of KCa channels by modulating their gating properties. Moreover, this review discusses the recent advances in our understanding of KCa channel assembly and anterograde trafficking toward the plasma membrane, the micro-domains in which these channels are expressed within the cell, and finally the retrograde trafficking routes these channels take following endocytosis. As the regulation of intracellular trafficking by agonists as well as the protein-protein interactions that modify these events continue to be explored, we anticipate this will open new therapeutic avenues for the targeting of these channels based on the pharmacological modulation of KCa channel density at the plasma membrane.

Dynamin- and Rab5-dependent endocytosis of a Ca2+ -activated K+ channel, KCa2.3.

Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca(2+)-activated K(+) channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co-localization, transmission electron microscopy and co-immunoprecipitation (co-IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co-localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co-IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA-mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane.

Characterization of the PCMBS-dependent modification of KCa3.1 channel gating.

Intermediate conductance, calcium-activated potassium channels are gated by the binding of intracellular Ca(2+) to calmodulin, a Ca(2+)-binding protein that is constitutively associated with the C terminus of the channel. Although previous studies indicated that the pore-lining residues along the C-terminal portion of S6 contribute to the activation mechanism, little is known about whether the nonluminal face of S6 contributes to this process. Here we demonstrate that the sulfhydral reagent, parachloromercuribenze sulfonate (PCMBS), modifies an endogenous cysteine residue predicted to have a nonluminal orientation (Cys(276)) along the sixth transmembrane segment (S6). Modification of Cys(276) manipulates the steady-state and kinetic behavior of the channel by shifting the gating equilibrium toward the open state, resulting in a left shift in apparent Ca(2+) affinity and a slowing in the deactivation process. Using a six-state gating scheme, our analysis shows that PCMBS slows the transition between the open state back to the third closed state. Interpreting this result in the context of the steady-state and kinetic data suggests that PCMBS functions to shift the gating equilibrium toward the open state by disrupting channel closing. In an attempt to understand whether the nonluminal face of S6 participates in the activation mechanism, we conducted a partial tryptophan scan of this region. Substituting a tryptophan for Leu(281) recapitulated the effect on the steady-state and kinetic behavior observed with PCMBS. Considering the predicted nonluminal orientation of Cys(276) and Leu(281), a simple physical interpretation of these results is that the nonluminal face of S6 forms a critical interaction surface mediating the transition into the closed conformation, suggesting the nonluminal C-terminal portion of S6 is allosterically coupled to the activation gate.

Immunofluorescence-based assay to identify modulators of the number of plasma membrane KCa3.1 channels.

BACKGROUND: Intermediate conductance Ca2+-dependent K+ channels (KCa3.1) have been proposed as therapeutic targets for numerous diseases. We recently characterized the endocytic fate of these channels; leading to the possibility that this can be pharmacologically manipulated, thereby altering the number of channels (N) at the plasma membrane. RESULTS & DISCUSSION: We demonstrate that plasma membrane-localized KCa3.1 can be rapidly(10 min) tagged with a fluorophore using a combination of a biotin ligase (BirA) acceptor peptide-tagged channel and an ER-localized BirA. Endocytosis of KCa3.1 was quantified using a 96-well plate format, demonstrating that the ubiquitin-activating enzyme E1 inhibitor UBEI-41, blocks the endocytosis of KCa3.1. CONCLUSION: We describe a novel method for identifying modulators of KCa endocytosis and demonstrate this can be used to modulate Nat the plasma membrane. It is anticipated that altering N will provide novel therapeutic strategies for targeting these channels in disease.

Role of S3 and S4 transmembrane domain charged amino acids in channel biogenesis and gating of KCa2.3 and KCa3.1.

The role of positively charged arginines in the fourth transmembrane domain (S4) and a single negatively charged amino acid in the third transmembrane domain (S3) on channel biogenesis and gating of voltage-gated K(+) channels (Kv) has been well established. Both intermediate (KCa3.1) and small (KCa2.x) conductance, Ca(2+)-activated K(+) channels have two conserved arginines in S4 and a single conserved glutamic acid in S3, although these channels are voltage-independent. We demonstrate that mutation of any of these charged amino acids in KCa3.1 or KCa2.3 to alanine, glutamine, or charge reversal mutations results in a rapid degradation (<30 min) of total protein, confirming the critical role of these amino acids in channel biogenesis. Mutation of the S4 arginine closest to the cytosolic side of KCa3.1 to histidine resulted in expression at the cell surface. Excised patch clamp experiments revealed that this Arg/His mutation had a dramatically reduced open probability (P(o)), relative to wild type channels. Additionally, we demonstrate, using a combination of short hairpin RNA, dominant negative, and co-immunoprecipitation studies, that both KCa3.1 and KCa2.3 are translocated out of the endoplasmic reticulum associated with Derlin-1. These misfolded channels are poly-ubiquitylated, recognized by p97, and targeted for proteasomal degradation. Our results suggest that S3 and S4 charged amino acids play an evolutionarily conserved role in the biogenesis and gating of KCa channels. Furthermore, these improperly folded K(+) channels are translocated out of the endoplasmic reticulum in a Derlin-1- and p97-dependent fashion, poly-ubiquitylated, and targeted for proteasomal degradation.