Differential association of the auxiliary subunit Kvbeta2 with Kv1.4 and Kv4.3 K+ channels

Kvbeta2 subunits associate with Kv1 and Kv4 K+ channels, but the basis of preferential association is not understood. For example, detergent resistance suggests stronger auxiliary subunit association with Kv4.2 than with Kv1.2, but Kvbeta2 preferentially localizes with the latter channels in brain. Here we examine the interaction of Kvbeta2 with two native binding partners in brain: Kv4.3 and Kv1.4. We show that the auxiliary subunit binds more efficiently to Kv1.4 than to Kv4.3 in mammalian cells. However, preexisting Kvbeta2 complexes with Kv1.4 and Kv4.3 have similar detergent sensitivity. Thus, preferential steady state binding may reflect a difference in initial association rather than stability. We also find that that the cytoplasmic C-terminus of Kv4.3 inhibits Kvbeta2 association. Apparently, a region proximal to the Kv4.3 pore contributes to the inefficient auxiliary subunit interaction that produces preferential binding of Kvbeta2 to Kv1 channels.     

Anorexic effect of K+ channel blockade in mesenteric arterial smooth muscle and intestinal epithelial cells.

Activity of voltage-gated K+ (Kv) channels controls membrane potential (E(m)). Membrane depolarization due to blockade of K+ channels in mesenteric artery smooth muscle cells (MASMC) should increase cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and cause vasoconstriction, which may subsequently reduce the mesenteric blood flow and inhibit the transportation of absorbed nutrients to the liver and adipose tissue. In this study, we characterized and compared the electrophysiological properties and molecular identities of Kv channels and examined the role of Kv channel function in regulating E(m) in MASMC and intestinal epithelial cells (IEC). MASMC and IEC functionally expressed multiple Kv channel alpha- and beta-subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3, and Kv9.3, as well as Kvbeta1.1, Kvbeta2.1, and Kvbeta3), but only MASMC expressed voltage-dependent Ca2+ channels. The current density and the activation and inactivation kinetics of whole cell Kv currents were similar in MASMC and IEC. Extracellular application of 4-aminopyridine (4-AP), a Kv-channel blocker, reduced whole cell Kv currents and caused E(m) depolarization in both MASMC and IEC. The 4-AP-induced E(m) depolarization increased [Ca2+]cyt in MASMC and caused mesenteric vasoconstriction. Furthermore, ingestion of 4-AP significantly reduced the weight gain in rats. These results suggest that MASMC and IEC express multiple Kv channel alpha- and beta-subunits. The function of these Kv channels plays an important role in controlling E(m). The membrane depolarization-mediated increase in [Ca2+]cyt in MASMC and mesenteric vasoconstriction may inhibit transportation of absorbed nutrients via mesenteric circulation and limit weight gain.     

Phosphorylation is required for alteration of kv1.5 K(+) channel function by the Kvbeta1.3 subunit.

The Kv1.5 K(+) channel is functionally altered by coassembly with the Kvbeta1.3 subunit, which induces fast inactivation and a hyperpolarizing shift in the activation curve. Here we examine kinase regulation of Kv1.5/Kvbeta1.3 interaction after coexpression in human embryonic kidney 293 cells. The protein kinase C inhibitor calphostin C (3 microM) removed the fast inactivation (66 +/- 1.9 versus 11 +/- 0.25%, steady state/peak current) and the beta-induced hyperpolarizing voltage shift in the activation midpoint (V(1/2)) (-21.9 +/- 1.4 versus -4.3 +/- 2.0 mV). Calphostin C had no effect on Kv1.5 alone with respect to inactivation kinetics and V(1/2). Okadaic acid, but not the inactive derivative, blunted both calphostin C effects (V(1/2) = -17.6 +/- 2.2 mV, 38 +/- 1.8% inactivation), consistent with dephosphorylation being required for calphostin C action. Calphostin C also removed the fast inactivation (57 +/- 2.6 versus 16 +/- 0.6%) and the shift in V(1/2) (-22.1 +/- 1.4 versus -2.1 +/- 2.0 mV) conferred onto Kv1.5 by the Kvbeta1.2 subunit, which shares only C terminus sequence identity with Kvbeta1. 3. In contrast, modulation of Kv1.5 by the Kvbeta2.1 subunit was unaffected by calphostin C. These data suggest that Kvbeta1.2 and Kvbeta1.3 subunit modification of Kv1.5 inactivation and voltage sensitivity require phosphorylation by protein kinase C or a related kinase.     

Somatic gene transfer of tagged K+ channel fragments to probe trafficking and electrical function in epithelial cells and cardiac myocytes

To evaluate the roles of the C-termini of K + channels in subcellular targeting and protein-protein interactions, we created fusion constructs of the cell-surface antigen CD8 and the C-termini of Kv4.3, Kv1.4 and KvLQT1. Using a Cre-lox recombination system, we made 3 adenoviruses containing a fusion of the N-terminal-and transmembrane segments of CD8 with the C-termini of each of the 3 K + channels. Expression in polarized Opossum Kidney (OK) epithelial cells led to localization of CD8-Kv4.3 and CD8-Kv1.4 into the apical and basolateral membranes, while CD8-KvLQT1 remained in the endoplasmic reticulum (ER), even when co-expressed with MinK. When expressed in rat cardiac myocytes in culture, all the 3 constructs were diffusely targeted to the surface membrane. The ER retention of CD8-KvLQT1 in OK cells but not in cardiomyocytes thus reveals functional differences in trafficking between these two cell types. To probe functional roles of C-termini, we studied K + currents in cardiac myocytes expressing CD8-Kv4.3. Patch-clamp recordings of transient outward current revealed a hyperpolarizing shift of steady-state inactivation, implying that CD8-Kv4.3 may be disrupting the interaction of Kv4.x channels with one or more as-yet-undefined regulatory subunits. Thus, expression of tagged ion-channel fragments represents a novel, generalizable approach that may help to elucidate assembly, localization and function of these important signaling proteins.     

Differential inhibition of transient outward currents of Kv1.4 and Kv4.3 by endothelin

The effects of endothelin on the transient outward K(+) currents were compared between Kv1.4 and Kv4.3 channels in Xenopus oocytes expression system. Both transient outward K(+) currents were decreased by stimulation of endothelin receptor ET(A) coexpressed with the K(+) channels. Transient outward current of Kv1.4 was decreased by about 85% after 10(-8) M ET-1, while that of Kv4.3 was decreased by about 60%. By mutagenesis experiments we identified two phosphorylation sites of PKC and CaMKII in Kv1.4 responsible for the decrease in I(to) by ET-1. In Kv4.3 a PKC phosphorylation site was identified which is in part responsible for the decrease in I(to). Differences in the suppression of I(to) could be ascribed to the difference in intracellular signaling including the number of phosphorylation sites. These findings might give clues for the understanding of molecular mechanism of ventricular arrhythmias in heart failure, in which endothelin is involved in the pathogenesis.     

Neonatal rat cardiac fibroblasts express three types of voltage-gated K+ channels: regulation of a transient outward current by protein kinase C

Cardiac fibroblasts regulate myocardial development via mechanical, chemical, and electrical interactions with associated cardiomyocytes. The goal of this study was to identify and characterize voltage-gated K(+) (Kv) channels in neonatal rat ventricular fibroblasts. With the use of the whole cell arrangement of the patch-clamp technique, three types of voltage-gated, outward K(+) currents were measured in the cultured fibroblasts. The majority of cells expressed a transient outward K(+) current (I(to)) that activated at potentials positive to -40 mV and partially inactivated during depolarizing voltage steps. I(to) was inhibited by the antiarrhythmic agent flecainide (100 microM) and BaCl(2) (1 mM) but was unaffected by 4-aminopyridine (4-AP; 0.5 and 1 mM). A smaller number of cells expressed one of two types of kinetically distinct, delayed-rectifier K(+) currents [I(K) fast (I(Kf)) and I(K) slow (I(Ks))] that were strongly blocked by 4-AP. Application of phorbol 12-myristate 13-acetate, to stimulate protein kinase C (PKC), inhibited I(to) but had no effect on I(Kf) and I(Ks). Immunoblot analysis revealed the presence of Kv1.4, Kv1.2, Kv1.5, and Kv2.1 alpha-subunits but not Kv4.2 or Kv1.6 alpha-subunits in the fibroblasts. Finally, pretreatment of the cells with 4-AP inhibited angiotensin II-induced intracellular Ca(2+) mobilization. Thus neonatal cardiac fibroblasts express at least three different Kv channels that may contribute to electrical/chemical signaling in these cells.     

Kvbeta subunits increase expression of Kv4.3 channels by interacting with their C termini

Auxiliary Kvbeta subunits form complexes with Kv1 family voltage-gated K(+) channels by binding to a part of the N terminus of channel polypeptide. This association influences expression and gating of these channels. Here we show that Kv4.3 proteins are associated with Kvbeta2 subunits in the brain. Expression of Kvbeta1 or Kvbeta2 subunits does not affect Kv4.3 channel gating but increases current density and protein expression. The increase in Kv4.3 protein is larger at longer times after transfection, suggesting that Kvbeta-associated channel proteins are more stable than those without the auxiliary subunits. This association between Kv4.3 and Kvbeta subunits requires the C terminus but not the N terminus of the channel polypeptide. Thus, Kvbeta subunits utilize diverse molecular interactions to stimulate the expression of Kv channels from different families.     

Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-dependent K(+) channels in neuroendocrine islet beta-cells through an interaction with the channel N terminus

Insulin secretion is initiated by ionic events involving membrane depolarization and Ca(2+) entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K(+) channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K(+) currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K(+) currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.     

Position-dependent attenuation by Kv1.6 of N-type inactivation of Kv1.4-containing channels

Assembly of distinct α subunits of Kv1 (voltage-gated K(+) channels) into tetramers underlies the diversity of their outward currents in neurons. Kv1.4-containing channels normally exhibit N-type rapid inactivation, mediated through an NIB (N-terminal inactivation ball); this can be over-ridden if associated with a Kv1.6 α subunit, via its NIP (N-type inactivation prevention) domain. Herein, NIP function was shown to require positioning of Kv1.6 adjacent to the Kv1.4 subunit. Using a recently devised gene concatenation, heterotetrameric Kv1 channels were expressed as single-chain proteins on the plasmalemma of HEK (human embryonic kidney)-293 cells, so their constituents could be arranged in different positions. Placing the Kv1.4 and 1.6 genes together, followed by two copies of Kv1.2, yielded a K(+) current devoid of fast inactivation. Mutation of critical glutamates within the NIP endowed rapid inactivation. Moreover, separating Kv1.4 and 1.6 with a copy of Kv1.2 gave a fast-inactivating K(+) current with steady-state inactivation shifted to more negative potentials and exhibiting slower recovery, correlating with similar inactivation kinetics seen for Kv1.4-(1.2)(3). Alternatively, separating Kv1.4 and 1.6 with two copies of Kv1.2 yielded slow-inactivating currents, because in this concatamer Kv1.4 and 1.6 should be together. These findings also confirm that the gene concatenation can generate K(+) channels with α subunits in pre-determined positions.     

Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five- fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage- gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.     

Differential modulation of Kv1 channel-mediated currents by co-expression of Kvbeta3 subunit in a mammalian cell-line

The effect of Kvbeta3 subunit co-expression on currents mediated by the Shaker-related channels Kv1.1 to Kv1.6 in Chinese hamster ovary (CHO) cells was studied with patch-clamp techniques. In the presence of Kvbeta3, differences in the voltage dependence of activation for Kv1.1, Kv1.3 and Kv1.6 were detected, but not for Kv1.2- and Kv1.4-mediated currents. Co-expression of Kvbeta3 did not cause a significant increase in current density for any of the tested channels. In contrast to previous studies in Xenopus oocyte expression system, Kvbeta3 confered a rapid inactivation to all except Kv1.3 channels. Also, Kv1.6 channels that possess an N-type inactivation prevention (NIP) domain for Kvbeta1.1, inactivated rapidly when co-expressed with Kvbeta3. Onset and recovery kinetics of channel inactivation distinctly differed for the various Kv1alpha/Kvbeta3 subunit combinations investigated in this study. The results indicate that the choice of expression system may critically determine Kvbeta3 inactivating activity. This suggests that the presence of an inactivating domain and a receptor in a channel pore, although necessary, may not be sufficient for an effective rapid N-type inactivation of Kv1 channels in heterologous expression systems.     

KCNE3 is an inhibitory subunit of the Kv4.3 potassium channel

The mammalian Kv4.3 potassium channel is a fast activating and inactivating K+ channel widely distributed in mammalian tissues. Kv4.3 is the major component of various physiologically important currents ranging from A-type currents in the CNS to the transient outward potassium conductance in the heart (I(to)). Here we show that the KCNE3 beta-subunit has a strong inhibitory effect on current conducted by heterologously expressed Kv4.3 channels. KCNE3 reduces the Kv4.3 current amplitude, and it slows down the channel activation and inactivation as well as the recovery from inactivation. KCNE3 also inhibits currents generated by Kv4.3 in complex with the accessory subunit KChIP2. We find the inhibitory effect of KCNE3 to be specific for Kv4.3 within the Kv4 channel family. Kv4.3 has previously been shown to interact with a number of beta-subunits, but none of the described subunit-interactions exert an inhibitory effect on the Kv4.3 current.     

NADPH binding to beta-subunit regulates inactivation of voltage-gated K(+) channels

Ancillary beta-subunits regulate the voltage-dependence and the kinetics of Kv currents. The Kvbeta proteins bind pyridine nucleotides with high affinity but the role of cofactor binding in regulating Kv currents remains unclear. We found that recombinant rat Kvbeta 1.3 binds NADPH (K(d)=1.8+/-0.02 microM) and NADP(+) (K(d)=5.5+/-0.9 microM). Site-specific modifications at Tyr-307 and Arg-316 decreased NADPH binding; whereas, K(d) NADPH was unaffected by the R241L mutation. COS-7 cells transfected with Kv1.5 cDNA displayed non-inactivating currents. Co-transfection with Kvbeta1.3 accelerated Kv activation and inactivation and induced a hyperpolarizing shift in voltage-dependence of activation. Kvbeta-mediated inactivation of Kv currents was prevented by the Y307F and R316E mutations but not by the R241L substitution. Additionally, the R316E mutation weakened Kvalpha-beta interaction. Inactivation of Kv currents by Kvbeta:R316E was restored when excess NADPH was included in the patch pipette. These observations suggest that NADPH binding is essential for optimal interaction between Kvalpha and beta subunits and for Kvbeta-induced inactivation of Kv currents.     

Inhibition of Kv2.1 voltage-dependent K+ channels in pancreatic beta-cells enhances glucose-dependent insulin secretion

Voltage-dependent (Kv) outward K(+) currents repolarize beta-cell action potentials during a glucose stimulus to limit Ca(2+) entry and insulin secretion. Dominant-negative "knockout" of Kv2 family channels enhances glucose-stimulated insulin secretion. Here we show that a putative Kv2.1 antagonist (C-1) stimulates insulin secretion from MIN6 insulinoma cells in a glucose- and dose-dependent manner while blocking voltage-dependent outward K(+) currents. C-1-blocked recombinant Kv2.1-mediated currents more specifically than currents mediated by Kv1, -3, and -4 family channels (Kv1.4, 3.1, 4.2). Additionally, C-1 had little effect on currents recorded from MIN6 cells expressing a dominant-negative Kv2.1 alpha-subunit. The insulinotropic effect of acute Kv2.1 inhibition resulted from enhanced membrane depolarization and augmented intracellular Ca(2+) responses to glucose. Immunohistochemical staining of mouse pancreas sections showed that expression of Kv2.1 correlated highly with insulin-containing beta-cells, consistent with the ability of C-1 to block voltage-dependent outward K(+) currents in isolated mouse beta-cells. Antagonism of Kv2.1 in an ex vivo perfused mouse pancreas model enhanced first- and second-phase insulin secretion, whereas glucagon secretion was unaffected. The present study demonstrates that Kv2.1 is an important component of beta-cell stimulus-secretion coupling, and a compound that enhances, but does not initiate, beta-cell electrical activity by acting on Kv2.1 would be a useful antidiabetic agent.     

Contributions of Kv1.2, Kv1.5 and Kv2.1 subunits to the native delayed rectifier K(+) current in rat mesenteric artery smooth muscle cells

A large array of voltage-gated K(+) channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 microg/ml) inhibited the Kv currents by 29.2 +/- 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 microg/ml) by 24.5 +/- 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4-20 microg/ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 microg/ml each) induced an additive inhibition of Kv currents by 42.3 +/- 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 microg/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.     

Kvbeta1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to kv4.2 but not to Shaker channels.

Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic modulatory beta subunits. The differential expression and association of alpha and beta subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells, and their functional expression in heterologous systems provides a tool to study their regulation at a molecular level. Here, we have studied the effects of Kvbeta1.2 coexpression on the properties of Shaker and Kv4.2 KV channel alpha subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kvbeta1.2 functionally associates with these two alpha subunits, as well as with the endogenous KV channels of HEK293 cells, to modulate different properties of the heteromultimers. Kvbeta1.2 accelerates the rate of inactivation of the Shaker currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kvbeta1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2'-dithiodipyridine), which also modulate the low pO2 response of the Kv4.2+beta channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the effects of DTT. Finally, hypoxic inhibition of Kv4.2+beta currents can be reverted by 70% in the presence of carbon monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that beta subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability of Kv4. 2+beta channels to respond to hypoxia.