Daniel C. Devor

Kinase-dependent Regulation of the Intermediate Conductance, Calcium-dependent Potassium Channel, hIK1

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca2+-dependent gating of the cloned intermediate conductance, Ca2+-dependent K+ channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca2+-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca2+-dependent increase in hIK1 activity (NPo). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg2+ and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI5–24. The Ca2+ dependence of hIK1 activation was best fit with a stimulatory constant (K s ) of 350 nm and a Hill coefficient (n) of 2.3. ATP increased NPo at [Ca2+] >100 nm while having no effect on K s or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg2+-dependent fashion, being reversed by alkaline phosphatase. Neither PKI5–24 nor CaMKII281–309 or PKC19–31 affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg2+- and PKI5–24-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII281–309 and PKC19–31 had no effect on the ATP-dependent activation. The Ca2+-dependent activation (K s and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca2+-dependent and occurs via an increase in NPo without an effect on either Ca2+ affinity or apparent cooperativity.

UTP inhibits Na+ absorption in wild-type and ΔF508 CFTR-expressing human bronchial epithelia

Ca2+-mediated agonists, including UTP, are being developed for therapeutic use in cystic fibrosis (CF) based on their ability to modulate alternative Cl- conductances. As CF is also characterized by hyperabsorption of Na+, we determined the effect of mucosal UTP on transepithelial Na+transport in primary cultures of human bronchial epithelia (HBE). In symmetrical NaCl, UTP induced an initial increase in short-circuit current ( I sc) followed by a sustained inhibition. To differentiate between effects on Na+ absorption and Cl- secretion, I sc was measured in the absence of mucosal and serosal Cl-( I Na). Again, mucosal UTP induced an initial increase and then a sustained decrease that reduced amiloride-sensitive I Na by 73%. The Ca2+-dependent agonists histamine, bradykinin, serosal UTP, and thapsigargin similarly induced sustained inhibition (62-84%) of I Na. Mucosal UTP induced similar sustained inhibition (half-maximal inhibitory concentration 296 nM) of I Na in primary cultures of human CF airway homozygous for the ΔF508 mutation. BAPTA-AM blunted UTP-dependent inhibition of I Na, but inhibitors of protein kinase C (PKC) and phospholipase A2 had no effect. Indeed, direct activation of PKC by phorbol 12-myristate 13-acetate failed to inhibit Na+ absorption. Apyrase, a tri- and diphosphatase, did not reverse inhibitory effects of UTP on I Na, suggesting a long-term inhibitory effect of UTP that is independent of receptor occupancy. After establishment of a mucosa-to-serosa K+ concentration gradient and permeabilization of the mucosal membrane with nystatin, mucosal UTP induced an initial increase in K+current followed by a sustained inhibition. We conclude that increasing cellular Ca2+ induces a long-term inhibition of transepithelial Na+transport across normal and CF HBE at least partly due to downregulation of a basolateral membrane K+ conductance. Thus UTP may have a dual therapeutic effect in CF airway: 1) stimulation of a Cl- secretory response and 2) inhibition of Na+ transport.

Modulation of K+ channels by arachidonic acid in T84 cells. I. Inhibition of the Ca2+-dependent K+ channel

The Cl- secretory response of colonic cells to Ca2+-mediated agonists is transient despite a sustained elevation of intracellular Ca2+. We evaluated the effects of second messengers proposed to limit Ca2+-mediated Cl- secretion on the basolateral membrane, Ca2+-dependent K+ channel (KCa) in colonic secretory cells, T84. Neither protein kinase C (PKC) nor inositol tetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affected KCa in excised inside-out patches. In contrast, arachidonic acid (AA; 3 μM) potently inhibited KCa, reducing NP o, the product of number of channels and channel open probability, by 95%. The apparent inhibition constant for this AA effect was 425 nM. AA inhibited KCa in the presence of both indomethacin and nordihydroguaiaretic acid, blockers of the cyclooxygenase and lipoxygenase pathways. In the presence of albumin, the effect of AA on KCa was reversed. A similar effect of AA was observed on KCa during outside-out recording. We determined also the effect of the cis-unsaturated fatty acid linoleate, the trans-unsaturated fatty acid elaidate, and the saturated fatty acid myristate. At 3 μM, all of these fatty acids inhibited KCa, reducing NP o by 72-86%. Finally, the effect of the cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3) on the carbachol-induced short-circuit current ( I sc) response was determined. In the presence of AACOCF3, the peak carbachol-induced I sc response was increased ∼2.5-fold. Our results suggest that AA generation induced by Ca2+-mediated agonists may contribute to the dissociation observed between the rise in intracellular Ca2+ evoked by these agonists and the associated Cl- secretory response.

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.—Balut, C. M., Loch, C. M., Devor, D. C. Role of ubiquitylation and USP8‐dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1. FASEB J. 25, 3938–3948 (2011). www.fasebj.org

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 Ca2+-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, Gly206–Pro217, as being required for the rapid recycling of KCa2.3. Deletion of Gly206–Pro217 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.

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, Ca2+-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 (Po), 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.

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.

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel

We used single-channel recording techniques to identify and characterize a large-conductance, Ca2+-independent K+ channel in the colonic secretory cell line T84. In symmetric potassium gluconate, this channel had a linear current-voltage relationship with a single-channel conductance of 161 pS. Channel open probability ( P o) was increased at depolarizing potentials. Partial substitution of bath K+ with Na+ indicated a permeability ratio of K+ to Na+ of 25:1. Channel P o was reduced by extracellular Ba2+. Event-duration analysis suggested a linear kinetic model for channel gating having a single open state and three closed states: C3←→C2←→C1←→O. Arachidonic acid (AA) increased the P o of the channel, with an apparent stimulatory constant ( K s) of 1.39 μM. Neither channel open time (O) nor the fast closed time (C1) was affected by AA. In contrast, AA dramatically reduced mean closed time by decreasing both C3 and C2. The cis-unsaturated fatty acid linoleate increased P oalso, whereas the saturated fatty acid myristate and the trans-unsaturated fatty acid elaidate did not affect P o. This channel is activated also by negative pressure applied to the pipette during inside-out recording. Thus we determined the effect of the stretch-activated channel blockers amiloride and Gd3+ on the K+ channel after activation by AA. Amiloride (2 mM) on the extracellular side reduced single-channel amplitude in a voltage-dependent manner, whereas Gd3+ (100 μM) had no effect on channel activity. Activation of this K+ channel may be important during stimulation of Cl- secretion by agonists that use AA as a second messenger (e.g., vasoactive intestinal polypeptide, adenosine) or during the volume regulatory response to cell swelling.

Trafficking of intermediate (KCa3.1) and small (KCa2.x) conductance, Ca2+-activated K+ channels: A novel target for medicinal chemistry efforts?

Ca2+‐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.

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.