PKC inhibition results in a Kv 1.5 + Kv ß1.3 pharmacology closer to Kv 1.5 channels.

BACKGROUND AND PURPOSE: The Kv ß1.3 subunit modifies the gating and pharmacology of Kv 1.5 channels in a PKC-dependent manner, decreasing channel sensitivity to bupivacaine- and quinidine-mediated blockade. Cardiac Kv 1.5 channels associate with receptor for activated C kinase 1 (RACK1), the Kv ß1.3 subunit and different PKC isoforms, resulting in the formation of a functional channelosome. The aim of the present study was to investigate the effects of PKC inhibition on bupivacaine and quinidine block of Kv 1.5 + Kv ß1.3 channels. EXPERIMENTAL APPROACH: HEK293 cells were transfected with Kv 1.5 + Kv ß1.3 channels, and currents were recorded using the whole-cell configuration of the patch-clamp technique. PKC inhibition was achieved by incubating the cells with either calphostin C or bisindolylmaleimide II and the effects of bupivacaine and quinidine were analysed. KEY RESULTS: The voltage-dependent inactivation of Kv 1.5 + Kv ß1.3 channels and their pharmacological behaviour after PKC inhibition with calphostin C were similar to those displayed by Kv 1.5 channels alone. Indeed, the IC50 values for bupivacaine were similar in cells whose PKC was inhibited with calphostin C or bisindolylmaleimide II. Similar results were also observed in the presence of quinidine. CONCLUSIONS AND IMPLICATIONS: The finding that the voltage-dependence of inactivation and the pharmacology of Kv 1.5 + Kv ß1.3 channels after PKC inhibition resembled that observed in Kv 1.5 channels suggests that both processes are dependent on PKC-mediated phosphorylation. These results may have clinical relevance in diseases that are characterized by alterations in kinase activity.

Different Kv2.1/Kv9.3 heteromer expression during brain and lung post-natal development in the rat.

The Kv2.1/Kv9.3 heteromer generates an O2 sensitive potassium channel and induces a slow deactivation that has important consequences for brain and lung physiology. We examined the developmental regulation of Kv2.1 and Kv9.3 mRNAs in brain and lung. Both genes followed parallel expression patterns in brain, increasing progressively through post-natal life. In lung, however, the expression of the two genes followed opposite trends: Kv2.1 transcripts decreased, while Kv9.3 mRNA increased. The Kv9.3/Kv2.1 ratio shows that while in brain the expression of both genes followed a similar pattern, the relative abundance of Kv9.3 increased steadily through post-natal life in lung. Furthermore, there is selective regulation of gene expression during the suckling-weaning transition. Our results suggest that different Kv9.3/Kv2.1 ratios could have physiological implications in both organs during post-natal development, and that diet composition and selective tissue-specific insulin regulation modulate the expression of Kv2.1 and Kv9.3.

Differential expression of K(V) channel alpha- and beta-subunits in the bovine pulmonary arterial circulation.

Resistance pulmonary arteries constrict in response to hypoxia, whereas conduit pulmonary arteries typically do not respond or dilate slightly. One proposed mechanism for this differential response is the variable expression of pulmonary arterial smooth muscle cell voltage-gated K(+) (K(V)) channel subunits (Kv1.2, Kv2.1, Kv1.5, and Kv3.1b) shown to be O(2) sensitive in heterologous expression systems. In this study, immunoblotting and immunohistochemistry were used to examine the expression of K(V) channel alpha- and beta-subunits in the bovine pulmonary arterial circulation to determine whether differential K(V) channel subunit distribution is responsible for the distinct sensitivities of pulmonary arteries to hypoxia. Surprisingly, there was little difference in the expression levels of Kv1.2, Kv1.5, and Kv2.1 between conduit and resistance pulmonary arteries. In contrast, expression of the Kv3.1b alpha-subunit and Kv beta.1, Kv beta 1.2, and Kv beta 1.3 accessory subunits dramatically increased along the pulmonary arterial tree. The differential expression of all the beta-subunits but of only one of the putative O(2)-sensitive alpha-subunits suggests that the alpha-subunits alone are not the O(2) sensors but further implicates the auxiliary beta-subunits in pulmonary arterial O(2) sensing.

Altered K(+) channel gene expression in diabetic rat ventricle: isoform switching between Kv4.2 and Kv1.4.

Expression of voltage-gated K(+) channels encoding the K(+) independent transient outward current in the streptozocin-induced diabetic (DM) rat ventricle was studied to determine the basis for slowed cardiac repolarization in diabetes mellitus. Although hypertrophy was not detected in diabetic rats at 12 wk after streptozocin treatment, ventricular Kv4.2 mRNA levels decreased 41% relative to nondiabetic controls. Kv1.4 mRNA levels increased 179% relative to controls, whereas Kv4.3 mRNA levels were unaffected. Immunohistochemistry and Western blot analysis of the diabetic heart showed that the density of the Kv4.2 protein decreased, whereas Kv1.4 protein increased. Thus isoform switching from Kv4.2 to Kv1.4 is most likely the mechanism underlying the slower kinetics of transient outward K(+) current observed in the diabetic ventricle. Brain Kv1.4, Kv4.2, or Kv4.3 mRNA levels were unaffected by diabetes. Myosin heavy chain (MHC) gene expression was altered with a 32% decrease in alpha-MHC mRNA and a 259% increase in beta-MHC mRNA levels in diabetic ventricle. Low-dose insulin-like growth factor-II (IGF-II) treatment during the last 6 of the 12 wk of diabetes (DM + IGF) protected against these changes in MHC mRNAs despite continued hyperglycemia and body weight loss. IGF-II treatment did not change K(+) channel mRNA levels in DM or control rat ventricles. Thus IGF treatment may prevent some, but not all, biochemical abnormalities in the diabetic heart.

Molecular basis of hypoxia-induced pulmonary vasoconstriction: role of voltage-gated K+ channels.

The hypoxia-induced membrane depolarization and subsequent constriction of small resistance pulmonary arteries occurs, in part, via inhibition of vascular smooth muscle cell voltage-gated K+ (KV) channels open at the resting membrane potential. Pulmonary arterial smooth muscle cell KV channel expression, antibody-based dissection of the pulmonary arterial smooth muscle cell K+ current, and the O2 sensitivity of cloned KV channels expressed in heterologous expression systems have all been examined to identify the molecular components of the pulmonary arterial O2-sensitive KV current. Likely components include Kv2.1/Kv9.3 and Kv1.2/Kv1.5 heteromeric channels and the Kv3.1b alpha-subunit. Although the mechanism of KV channel inhibition by hypoxia is unknown, it appears that KV alpha-subunits do not sense O2 directly. Rather, they are most likely inhibited through interaction with an unidentified O2 sensor and/or beta-subunit. This review summarizes the role of KV channels in hypoxic pulmonary vasoconstriction, the recent progress toward the identification of KV channel subunits involved in this response, and the possible mechanisms of KV channel regulation by hypoxia.

Isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations. Targeting of Kv1.5 to caveolae.

The precise subcellular localization of ion channels is often necessary to ensure rapid and efficient integration of both intracellular and extracellular signaling events. Recently, we have identified lipid raft association as a novel mechanism for the subcellular sorting of specific voltage-gated K(+) channels to regions of the membrane rich in signaling complexes. Here, we demonstrate isoform-specific targeting of voltage-gated K(+) (Kv) channels to distinct lipid raft populations with the finding that Kv1.5 specifically targets to caveolae. Multiple lines of evidence indicate that Kv1.5 and Kv2.1 exist in distinct raft domains: 1) channel/raft association shows differential sensitivity to increasing concentrations of Triton X-100; 2) unlike Kv2.1, Kv1.5 colocalizes with caveolin on the cell surface and redistributes with caveolin following microtubule disruption; and 3) immunoisolation of caveolae copurifies Kv1.5 channel. Both depletion of cellular cholesterol and inhibition of sphingolipid synthesis alter Kv1.5 channel function by inducing a hyperpolarizing shift in the voltage dependence of activation and inactivation. The differential targeting of Kv channel subtypes to caveolar and noncaveolar rafts within a single membrane represents a unique mechanism of compartmentalization, which may permit isoform-specific modulation of K(+) channel function.

Presence of the Kv1.5 K(+) channel in the sinoatrial node.

The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K(+) channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, approximately 64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gap junctions.

Effects of a quaternary bupivacaine derivative on delayed rectifier K(+) currents.

Block of hKv1.5 channels by R-bupivacaine has been attributed to the interaction of the charged form of the drug with an intracellular receptor. However, bupivacaine is present as a mixture of neutral and charged forms both extra- and intracellularly. We have studied the effects produced by the R(+) enantiomer of a quaternary bupivacaine derivative, N-methyl-bupivacaine, (RB(+)1C) on hKv1.5 channels stably expressed in Ltk(-) cells using the whole-cell configuration of the patch-clamp technique. When applied from the intracellular side of the membrane, RB(+)1C induced a time- and voltage-dependent block similar to that induced by R-bupivacaine. External application of 50 microM RB(+)1C reduced the current at +60 mV by 24+/-2% (n=10), but this block displayed neither time- nor voltage-dependence. External RB(+)1C partially relieved block induced by R-bupivacaine (61+/-2% vs 56+/-3%, n=4, P<0.05), but it did not relieve block induced by internal RB(+)1C. In addition, it did not induce use-dependent block, but when applied in combination with internal RB(+)1C a use-dependent block that increased with pulse duration was observed. These results indicate that RB(+)1C induces different effects on hKv1.5 channels when applied from the intra or the extracellular side of the membrane, suggesting that the actions of bupivacaine are the resulting of those induced on the external and the internal side of hKv1.5 channels.

Differential targeting of Shaker-like potassium channels to lipid rafts.

Ion channel targeting within neuronal and muscle membranes is an important determinant of electrical excitability. Recent evidence suggests that there exists within the membrane specialized microdomains commonly referred to as lipid rafts. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction proteins such as nitric-oxide synthase, ligand-gated receptors, and multiple protein kinases. Here, we demonstrate that the voltage-gated K(+) channel Kv2.1, but not Kv4.2, targets to lipid rafts in both heterologous expression systems and rat brain. The Kv2.1 association with lipid rafts does not appear to involve caveolin. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1 associated rafts and shifts the midpoint of Kv2.1 inactivation by nearly 40 mV without affecting peak current density or channel activation. The differential targeting of Kv channels to lipid rafts represents a novel mechanism both for the subcellular sorting of K(+) channels to regions of the membrane rich in signaling complexes and for modulating channel properties via alterations in lipid content.

Oxygen sensitivity of cloned voltage-gated K(+) channels expressed in the pulmonary vasculature.

Hypoxic pulmonary vasoconstriction is initiated by inhibiting one or more voltage-gated potassium (Kv) channel in the vascular smooth muscle cells (VSMCs) of the small pulmonary resistance vessels. Although progress has been made in identifying which Kv channel proteins are expressed in pulmonary arterial (PA) VSMCs, there are conflicting reports regarding which channels contribute to the native O(2)-sensitive K(+) current. In this study, we examined the effects of hypoxia on the Kv1.2, Kv1.5, Kv2.1, and Kv9.3 alpha subunits expressed in mouse L cells using the whole-cell patch-clamp technique. Hypoxia (PO(2)= approximately 30 mm Hg) reversibly inhibited Kv1.2 and Kv2.1 currents only at potentials more positive than 30 mV. In contrast, hypoxia did not alter Kv1.5 current. Currents generated by coexpression of Kv2.1 with Kv9.3 alpha subunits were reversibly inhibited by hypoxia in the voltage range of the resting membrane potential (E(M)) of PA VSMCs ( approximately 28% at -40 mV). Coexpression of Kv1.2 and Kv1.5 alpha subunits produced currents that displayed kinetic and pharmacological properties distinct from Kv1.2 and Kv1.5 channels expressed alone. Moreover, hypoxia reversibly inhibited Kv1.2/Kv1.5 current activated at physiologically relevant membrane potentials ( approximately 65% at -40 mV). These results indicate that (1) hypoxia reversibly inhibits Kv1.2 and Kv2.1 but not Kv1.5 homomeric channels, (2) Kv1.2 and 1.5 alpha subunits can assemble to form an O(2)-sensitive heteromeric channel, and (3) only Kv1.2/Kv1.5 and Kv2.1/Kv9.3 heteromeric channels are inhibited by hypoxia in the voltage range of the PA VSMC E(M). Thus, these heteromeric channels are strong candidates for the K(+) channel isoforms initiating hypoxic pulmonary vasoconstriction.

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.

Protein kinase A phosphorylation alters Kvbeta1.3 subunit-mediated inactivation of the Kv1.5 potassium channel.

The human Kv1.5 potassium channel forms the IKur current in atrial myocytes and is functionally altered by coexpression with Kvbeta subunits. To explore the role of protein kinase A (PKA) phosphorylation in beta-subunit function, we examined the effect of PKA stimulation on Kv1.5 current following coexpression with either Kvbeta1.2 or Kvbeta1.3, both of which coassemble with Kv1.5 and induce fast inactivation. In Xenopus oocytes expressing Kv1.5 and Kvbeta1.3, activation of PKA reduced macroscopic inactivation with an increase in K+ current. Similar results were obtained using HEK 293 cells which lack endogenous K+ channel subunits. These effects did not occur when Kv1.5 was coexpressed with either Kvbeta1.2 or Kvbeta1.3 lacking the amino terminus, suggesting involvement of this region of Kvbeta1.3. Removal of a consensus PKA phosphorylation site on the Kvbeta1.3 NH2 terminus (serine 24), but not alternative sites in either Kvbeta1.3 or Kv1.5, resulted in loss of the functional effects of kinase activation. The effects of phosphorylation appeared to be electrostatic, as replacement of serine 24 with a negatively charged amino acid reduced beta-mediated inactivation, while substitution with a positively charged residue enhanced it. These results indicate that Kvbeta1.3-induced inactivation is reduced by PKA activation, and that phosphorylation of serine 24 in the subunit NH2 terminus is responsible.

Functional expression of an inactivating potassium channel (Kv4.3) in a mammalian cell line.

OBJECTIVE: The goal of this study was to characterize the electrophysiological properties of the Kv4.3 channels expressed in a mammalian cell line. METHODS: Currents were recorded using the whole-cell voltage clamp technique. RESULTS: The threshold for activation of the expressed Kv4.3 current was approximately -30 mV. The dominant time constant for activation was 1.71 +/- 0.16 ms (n = 10) at +60 mV. The current inactivated, this process being incomplete, resulting in a sustained level which contributed 15 +/- 2% (n = 25) of the total current. The time course of inactivation was fit by a biexponential function, the fast component contributing 74 +/- 5% (n = 9) to the overall inactivation. The fast time constant was voltage-dependent [27.6 +/- 2.0 ms at +60 mV (n = 10) versus 64.0 +/- 3.6 ms at 0 mV (n = 10); P < 0.01], whereas the slow was voltage-independent [142 +/- 15 ms at +60 mV (n = 10) versus 129 +/- 33 ms at 0 mV (n = 6) P > 0.05]. The voltage-dependence of inactivation exhibited midpoint and slope values of -26.9 +/- 1.5 mV and 5.9 +/- 0.3 mV (n = 21). Recovery from inactivation was faster at more negative membrane potentials [203 +/- 17 ms (n = 13) and 170 +/- 19 ms (n = 4), at -90 and -100 mV]. Bupivacaine block of Kv4.3 channels was not stereoselective (KD approximately 31 microM). CONCLUSIONS: The functional profile of Kv4.3 channels expressed in Ltk- cells corresponds closely to rat ITO, although differences in recovery do not rule out association with accessory subunits. Nevertheless, the sustained component needs to be considered with respect to native ITO.

Modulation of Kv channel alpha/beta subunit interactions.

Voltage-gated K(+) channels comprise the largest and most diverse class of ion channels. These channels establish the resting membrane potential and modulate the frequency and duration of action potentials in nerve and muscle, as well as being the targets of several antiarrhythmic drugs in the heart. The multiplicity of Kv channel function is further enhanced through modulation by accessory beta subunits, which confer rapid inactivation, alter current amplitudes, and promote cell surface expression. In addition, alpha/beta interactions are also influenced by second messenger pathways. Recent evidence demonstrates that phosphorylation of Kv channel alpha and/or beta subunits may dramatically affect channel properties. The functional response of different K(+) channel subunits to activation of protein kinases represents not only a means to modulate subunit interactions, but also another mechanism for K(+) channel diversity in vivo.

Distinct domains of the voltage-gated K+ channel Kv beta 1.3 beta-subunit affect voltage-dependent gating.

The Kvbeta1.3 subunit confers a voltage-dependent, partial inactivation (time constant = 5.76 +/- 0.14 ms at +50 mV), an enhanced slow inactivation, a hyperpolarizing shift in the activation midpoint, and an increase in the deactivation time constant of the Kv1.5 delayed rectifier. Removal of the first 10 amino acids from Kvbeta1.3 eliminated the effects on fast and slow inactivation but not the voltage shift in activation. Addition of the first 87 amino acids of Kvbeta1.3 to the amino terminus of Kv1.5 reconstituted fast and slow inactivation without altering the midpoint of activation. Although an internal pore mutation that alters quinidine block (V512A) did not affect Kvbeta1.3-mediated inactivation, a mutation of the external mouth of the pore (R485Y) increased the extent of fast inactivation while preventing the enhancement of slow inactivation. These data suggest that 1) Kvbeta1.3-mediated effects involve at least two distinct domains of this beta-subunit, 2) inactivation involves open channel block that is allosterically linked to the external pore, and 3) the Kvbeta1.3-induced shift in the activation midpoint is functionally distinct from inactivation.

Molecular determinants of stereoselective bupivacaine block of hKv1.5 channels.

Enantiomers of local anesthetics are useful probes of ion channel structure that can reveal three-dimensional relations for drug binding in the channel pore and may have important clinical consequences. Bupivacaine block of open hKv1.5 channels is stereoselective, with the R(+)-enantiomer being 7-fold more potent than the S(-)-enantiomer (Kd = 4.1 mumol/L versus 27.3 mumol/L). Using whole-cell voltage clamp of hKv1.5 channels and site-directed mutants stably expressed in Ltk- cells, we have identified a set of amino acids that determine the stereoselectivity of bupivacaine block. Replacement of threonine 505 by hydrophobic amino acids (isoleucine, valine, or alanine) abolished stereoselective block, whereas a serine substitution preserved it [Kd = 60 mumol/L and 7.4 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. A similar substitution at the internal tetraethylammonium binding site (T477S) reduced the affinity for both enantiomers similarly, thus preserving the stereoselectivity [Kd = 45.5 mumol/L and 7.8 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. Replacement of L508 or V512 by a methionine (L508M and V512M) abolished stereoselective block, whereas substitution of V512 by an alanine (V512A) preserved it. Block of Kv2.1 channels, which carry valine, leucine, and isoleucine residues at T505, L508, and V512 equivalent sites, respectively, was not stereoselective [Kd = 8.3 mumol/L and 13 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. These results suggest that (1) the bupivacaine binding site is located in the inner mouth of the pore, (2) stereoselective block displays subfamily selectivity, and (3) a polar interaction with T505 combined with hydrophobic interactions with L508 and V512 are required for stereoselective block.

Molecular analysis of a binding site for quinidine in a human cardiac delayed rectifier K+ channel. Role of S6 in antiarrhythmic drug binding.

The antiarrhythmic agent quinidine blocks the human cardiac hKv1.5 channel expressed in mammalian cells at therapeutically relevant concentrations (EC50, 6.2 mumol/L). Mechanistic analysis has suggested that quinidine acts as a cationic open-channel blocker at a site in the internal mouth of the ionic pore and that binding is stabilized by hydrophobic interactions. We tested these hypotheses using site-directed mutagenesis of residues proposed to line the internal mouth of the channel or of nearby residues. Amino acid substitutions in the midsection of S6 (T505I, T505V, T505S, and V512A) reduced the dissociation rate for quinidine, increased the affinity (0.7, 1.5, 3.4, and 1.4 mumol/L, respectively), and preserved both the voltage-dependent open channel-block mechanism and the electrical binding distance (0.19 to 0.22). In contrast, smaller or nonsignificant effects were observed for: deletion of the intracellular C-terminal domain, charge neutralizations in the region immediately C-terminal to S6, elimination of aromatic residues in S6, and mutations at the putative internal turn of the P loop, at the external entrance of the pore, and at sites in the S4S5 linker. The approximately 10-fold increase in affinity with T505I and the reduction of the dissociation rate constant with the mutations that increased affinity are consistent with a hydrophobic stabilization of binding. Moreover, the T505 and V512 residues align on the same side of the putative alpha-helical S6 segment. Taken together, these results localize the hydrophobic binding site for this antiarrhythmic drug in the internal mouth of this human K+ channel and provide molecular support for the open channel-block model and the role of S6 in contributing to the inner pore.

Immunolocalization of the mNav2.3 Na+ channel in mouse heart: upregulation in myometrium during pregnancy.

mNav2.3 is a putative voltage-dependent sodium channel (NaC) gene expressed in both mouse heart and uterus that shares only 45% amino acid identity with NaCs from gene subfamily 1. Immunofluorescence studies using polyclonal antibodies against two distinct epitopes revealed that mNav2.3 protein in heart colocalized with nerve-specific antibody binding. Similar mNav2.3-specific antibody staining was observed in virgin uterus. However, mNav2.3 expression in uterine nerve disappeared during late pregnancy, concurrent with an appearance in both the longitudinal and circular uterine smooth muscle, which reached a maximum at term and quickly declined within 2 days postpartum. mNav2.3 expression in term uterus often colocalized on the myocyte surface with connexin 43. The immunofluorescence results are supported by Western analysis in which the 217-kDa NaC increased during late pregnancy and declined 2 days postpartum. These data provide perhaps the most dramatic example of NaC regulation. The acute and transient upregulation in myometrium during gestation suggests the Nav2.3 channel plays a role in uterine function at term.

Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kv beta 2.1 subunits.

The voltage-sensitive currents observed following hKv1.5 alpha subunit expression in HEK 293 and mouse L-cells differ in the kinetics and voltage dependence of activation and slow inactivation. Molecular cloning, immunopurification, and Western blot analysis demonstrated that an endogenous L-cell Kv beta 2.1 subunit assembled with transfected hKv 1.5 protein. In contrast, both mRNA and protein analysis failed to detect a beta subunit in the HEK 293 cells, suggesting that functional differences observed between these two systems are due to endogenous L-cell Kv beta 2.1 expression. In the absence of Kv beta 2.1, midpoints for activation and inactivation of hKv1.5 in HEK 293 cells were -0.2 +/- 2.0 and -9.6 +/- 1.8 mV, respectively. In the presence of Kv beta 2.1 these values were -14.1 +/- 1.8 and -22.1 +/- 3.7 mV, respectively. The beta subunit also caused a 1.5-fold increase in the extent of slow inactivation at 50 mV, thus completely reconstituting the L-cell current phenotype in the HEK 293 cells. These results indicate that 1) the Kv beta 2.1 subunit can alter Kv1.5 alpha subunit function, 2) beta subunits are not required for alpha subunit expression, and 3) endogenous beta subunits are expressed in heterologous expression systems used to study K+ channel function.