A novel toxin from the venom of the scorpion Tityus trivittatus, is the first member of a new alpha-KTX subfamily

The first example of a new sub-family of toxins (alpha-KTx20.1) from the scorpion Tityus trivittatus was purified, sequenced and characterized physiologically. It has 29 amino acid residues, three disulfide bridges assumed to adopt the cysteine-stabilized alpha/beta scaffold with a pI value of 8.98. The sequence identities with all the other known alpha-KTx are less than 40%. Its effects were verified using seven different cloned K(+) channels (vertebrate Kv1.1-1.5, Shaker IR and hERG) expressed in Xenopus leavis oocytes. The toxin-induced effects show large differences among the different K(+) channels and a preference towards Kv1.3 (EC50=7.9+/-1.4 nM).     

The first potassium channel toxin from the venom of the Iranian scorpion Odonthobuthus doriae

The very first member of K(+) channels toxins from the venom of the Iranian scorpion Odonthobuthus doriae (OdK1) was purified, sequenced and characterized physiologically. OdK1 has 29 amino acids, six conserved cysteines and a pI value of 4.95. Based on multiple sequence alignments, OdK1 was classified as alpha-KTx 8.5. The pharmacological effects of OdK1 were studied on six different cloned K(+) channels (vertebrate Kv1.1-Kv1.5 and Shaker IR) expressed in Xenopus laevis oocytes. Interestingly, OdK1 selectively inhibited the currents through Kv1.2 channels with an IC50 value of 183+/-3 nM but did not affect any of the other channels.     

The pore region of the Kv1.2alpha subunit is an important component of recombinant Kv1.2 channel oxygen sensitivity

Oxygen-sensitive K(+) channels are important elements in the cellular response to hypoxia. Although much progress has been made in identifying their molecular composition, the structural components associated to their O(2)-sensitivity are not yet understood. Recombinant Kv1.2 currents expressed in Xenopus oocytes are inhibited by a decrease in O(2) availability. On the contrary, heterologous Kv2.1 channels are O(2)-insensitive. To elucidate the protein segment responsible for the O(2)-sensitivity of Kv1.2 channels, we analyzed the response to anoxia of Kv1.2/Kv2.1 chimeric channels. Expression of chimeric Kv2.1 channels each containing the S4, the S1-S3 or the S6-COOH segments of Kv1.2 polypeptide resulted in a K(+) current insensitive to anoxia. In contrast, transferring the S5-S6 segment of Kv1.2 into Kv2.1 produced an O(2)-sensitive K(+) current. Finally, mutating a redox-sensitive methionine residue (M380) of Kv1.2 polypeptide did not affect O(2)-sensitivity. Thus, the pore and its surrounding regions of Kv1.2 polypeptide confer its hypoxic inhibition. This response is independent on the redox modulation of methionine residues in this protein segment.     

Identification of two types of ATP-sensitive K+ channels in rat ventricular myocytes

The ATP-sensitive K(+) (K(ATP)) channels are known to provide a functional linkage between the electrical activity of the cell membrane and metabolism. Two types of inwardly rectifying K(+) channel subunits (i.e., Kir6.1 and Kir6.2) with which sulfonylurea receptors are associated were reported to constitute the K(ATP) channels. In this study, we provide evidence to show two types of K(ATP) channels with different biophysical properties functionally expressed in isolated rat ventricular myocytes. Using patch-clamp technique, we found that single-channel conductance for the different two types of K(ATP) channels in these cells was 57 and 21 pS. The kinetic properties, including mean open time and bursting kinetics, did not differ between these two types of K(ATP) channels. Diazoxide only activated the small-conductance K(ATP) channel, while pinacidil and dinitrophenol stimulated both channels. Both of these K(ATP) channels were sensitive to block by glibenclamide. Additionally, western blotting, immunochemistry, and RT-PCR revealed two types of Kir6.X channels, i.e., Kir6.1 and Kir6.2, in rat ventricular myocytes. Single-cell Ca(2+) imaging also revealed that similar to dinitrophenol, diazoxide reduced the concentration of intracellular Ca(2+). The present results suggest that these two types of K(ATP) channels may functionally be related to the activity of heart cells.     

Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.     

Synthesis, 3-D structure, and pharmacology of a reticulated chimeric peptide derived from maurotoxin and Tsk scorpion toxins

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulfide bridges that acts on both Ca(2+)-activated (SK) and voltage-gated (Kv) K(+) channels. A 38-mer chimera of MTX, Tsk-MTX, has been synthesized by the solid-phase method. It encompasses residues from 1 to 6 of Tsk at N-terminal, and residues from 3 to 34 of MTX at C-terminal. As established by enzyme cleavage, Tsk-MTX displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7 and C4-C8 which, contrary to MTX, correspond to a disulfide bridge pattern common to known scorpion toxins. The 3-D structure of Tsk-MTX, solved by (1)H NMR, demonstrates that it adopts the alpha/beta scaffold of scorpion toxins. In vivo, Tsk-MTX is lethal by intracerebroventricular injection in mice (LD(50) value of 0.2 microg/mouse). In vitro, Tsk-MTX is as potent as MTX, or Tsk, to interact with apamin-sensitive SK channels of rat brain synaptosomes (IC(50) value of 2.5 nM). It also blocks voltage-gated K(+) channels expressed in Xenopus oocytes, but is inactive on rat Kv1.3 contrary to MTX.     

Permeation properties of inward-rectifier potassium channels and their molecular determinants

The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K(+) channels ROMK2 (Kir1.1b), IRK1 (Kir2.1), and their chimeras using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode with different permeant cations in the pipette. For inward K(+) conduction, replacing the extracellular loop of ROMK2 with that of IRK1 increased single-channel conductance by 25 pS (from 39 to 63 pS), whereas replacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance by 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH(2) terminus or transmembrane domains, suggesting that the two domains form two resistors in series. The larger conductance of the extracellular loop of IRK1 was attributable to a single amino acid difference (Thr versus Val) at the 3P position, three residues in front of the GYG motif. Permeability sequences for the conducted ions were similar for the two channels: Tl(+) > K(+) > Rb(+) > NH(4)(+). The ion selectivity sequence for ROMK2 based on conductance ratios was NH(4)(+) (1.6) > K(+) (1) > Tl(+) (0.5) > Rb(+) (0.4). For IRK1, the sequence was K(+) (1) > Tl(+) (0.8) > NH(4)(+) (0.6) > Rb(+) (0.1). The difference in the NH(4)(+)/ K(+) conductance (1.6) and permeability (0.09) ratios can be explained if NH(4)(+) binds with lower affinity than K(+) to sites within the pore. The relatively low conductances of NH(4)(+) and Rb(+) through IRK1 were again attributable to the 3P position within the P region. Site-directed mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred the relatively high Tl(+) conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectivity properties of the pore.     

Modulation of the Shaker K(+) channel gating kinetics by the S3-S4 linker

In Shaker K(+) channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3-S4 linker. To investigate the role of the S3-S4 linker in the ShakerH4Delta(6-46) (ShakerDelta) K(+) channel activation, we constructed S3-S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K(+) currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerDelta. The apparent number of gating charges was 12.6+/-0.6 e(o) in ShakerDelta, 12.7+/-0.5 in 10 aa linker, and 12.3+/-0.9 in 5 aa linker channels, but it was only 5.6+/-0.3 e(o) in the 0 aa linker mutant channel. The maximum probability of opening (P(o)(max)) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89x and 45x slower than that of the ShakerDelta K(+) channel, respectively. The initial lag of ionic currents when the prepulse was varied from -130 to -60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3-S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3-S4 linker plays an important role in setting ShakerDelta channel activation and deactivation kinetics.     

Intracellular trafficking of FXYD1 (phospholemman) and FXYD7 proteins in Xenopus oocytes and mammalian cells

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.     

Isolation of the first toxin from the scorpion Buthus occitanus israelis showing preference for Shaker potassium channels

We have purified BoiTx1, the first toxin from the venom of the Israeli scorpion, Buthus occitanus israelis, and studied its activity and genomic organization. BoiTx1 is a 37 amino acid-long peptide contained six conserved cysteines, and is classified as an alpha-KTx3.10 toxin. The pharmacological effects of BoiTx1 were studied on various cloned K(+) channels expressed in Xenopus laevis oocytes. BoiTx1 inhibited currents through Drosophila Shaker channels with an IC(50) value of 3.5+/-0.5nM, yet had much lesser effect on its mammalian orthologs. Thus, BoiTx1 is the first member of the alpha-KTx3 family that preferentially affects insect potassium channels.     

Uncoupling of gating charge movement and closure of the ion pore during recovery from inactivation in the Kv1.5 channel

Both wild-type (WT) and nonconducting W472F mutant (NCM) Kv1.5 channels are able to conduct Na(+) in their inactivated states when K(+) is absent. Replacement of K(+) with Na(+) or NMG(+) allows rapid and complete inactivation in both WT and W472F mutant channels upon depolarization, and on return to negative potentials, transition of inactivated channels to closed-inactivated states is the first step in the recovery of the channels from inactivation. The time constant for immobilized gating charge recovery at -100 mV was 11.1 +/- 0.4 ms (n = 10) and increased to 19.0 +/- 1.6 ms (n = 3) when NMG(+)(o) was replaced by Na(+)(o). However, the decay of the Na(+) tail currents through inactivated channels at -100 mV had a time constant of 129 +/- 26 ms (n = 18), much slower than the time required for gating charge recovery. Further experiments revealed that the voltage-dependence of gating charge recovery and of the decay of Na(+) tail currents did not match over a 60 mV range of repolarization potentials. A faster recovery of gating charge than pore closure was also observed in WT Kv1.5 channels. These results provide evidence that the recovery of the gating elements is uncoupled from that of the pore in Na(+)-conducting inactivated channels. The dissociation of the gating charge movements and the pore closure could also be observed in the presence of symmetrical Na(+) but not symmetrical Cs(+). This difference probably stems from the difference in the respective abilities of the two ions to limit inactivation to the P-type state or prevent it altogether.     

Role for calcium in the development of ovarial patency in Heliothis virescens

Insect oocytes sequester nutritive proteins from the hemolymph under the regulation by juvenile hormone (JH), in a process called patency. Here, a pharmacological approach was used to decipher the role for calcium in ovarial patency in the moth, Heliothis virescens. Follicular epithelial cells were exposed in calcium-free or calcium-containing media to JH I, JH II or JH III alone, or in combination with various inhibitors of signal transduction. Protein kinase inhibitors, Na(+)/K(+) -ATPase inhibitor, ouabain, an inhibitor of voltage-dependent calcium channels in plasma membrane, omega-Conotoxin MVII, endoplasmic reticulum (ER) Ca(2+) -ATPase inhibitor, thapsigargin, ER inositol 1,4,5-triphosphate receptor (IP(3)R) inhibitor, 2-ABP and ER ryanodine receptor (RyR) inhibitor, ryanodine, were used. The results of our study suggest that JH II evokes patency via protein kinase C-dependent signaling pathway, and activation of Na(+)/K(+) -ATPase, similar to JH III. Response to JH II and JH III predominantly relies upon external and internal calcium stores, using voltage-dependent calcium channels, IP(3)Rs and RyRs. In contrast, regulation of patency by JH I appears to be largely calcium independent, and the calcium-dependent component of the signaling pathway likely does not use IP(3)Rs, but RyRs only. The JH II, JH III and calcium-dependent component of JH I signaling pathway probably utilize calcium/calmodulin-dependent kinase II for activation of Na(+)/K(+) -ATPase.     

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

Rapid induction of P/C-type inactivation is the mechanism for acid-induced K+ current inhibition

Extracellular acidification is known to decrease the conductance of many voltage-gated potassium channels. In the present study, we investigated the mechanism of H(+)(o)-induced current inhibition by taking advantage of Na(+) permeation through inactivated channels. In hKv1.5, H(+)(o) inhibited open-state Na(+) current with a similar potency to K(+) current, but had little effect on the amplitude of inactivated-state Na(+) current. In support of inactivation as the mechanism for the current reduction, Na(+) current through noninactivating hKv1.5-R487V channels was not affected by [H(+)(o)]. At pH 6.4, channels were maximally inactivated as soon as sufficient time was given to allow activation, which suggested two possibilities for the mechanism of action of H(+)(o). These were that inactivation of channels in early closed states occurred while hyperpolarized during exposure to acid pH (closed-state inactivation) and/or inactivation from the open state was greatly accelerated at low pH. The absence of outward Na(+) currents but the maintained presence of slow Na(+) tail currents, combined with changes in the Na(+) tail current time course at pH 6.4, led us to favor the hypothesis that a reduction in the activation energy for the inactivation transition from the open state underlies the inhibition of hKv1.5 Na(+) current at low pH.     

Collapse of conductance is prevented by a glutamate residue conserved in voltage-dependent K(+) channels

Voltage-dependent K(+) channel gating is influenced by the permeating ions. Extracellular K(+) determines the occupation of sites in the channels where the cation interferes with the motion of the gates. When external [K(+)] decreases, some K(+) channels open too briefly to allow the conduction of measurable current. Given that extracellular K(+) is normally low, we have studied if negatively charged amino acids in the extracellular loops of Shaker K(+) channels contribute to increase the local [K(+)]. Surprisingly, neutralization of the charge of most acidic residues has minor effects on gating. However, a glutamate residue (E418) located at the external end of the membrane spanning segment S5 is absolutely required for keeping channels active at the normal external [K(+)]. E418 is conserved in all families of voltage-dependent K(+) channels. Although the channel mutant E418Q has kinetic properties resembling those produced by removal of K(+) from the pore, it seems that E418 is not simply concentrating cations near the channel mouth, but has a direct and critical role in gating. Our data suggest that E418 contributes to stabilize the S4 voltage sensor in the depolarized position, thus permitting maintenance of the channel open conformation.     

High glucose protects single beating adult cardiomyocytes against hypoxia

In the heart, the opening of sarcolemmal ATP-sensitive K(+) (K(ATP)) channels seems to be crucial for the cardiac protection against hypoxia/ischaemia. In the present study, we have exposed cardiomyocytes under hypoxia to high extracellular glucose (30 mM). Under these conditions, intracellular concentration of 1,3-bisphosphoglycerate has increased confirming stimulation of glycolysis. Perforated patch-clamp electrophysiology revealed that hypoxia induces whole-cell K(+) current in cardiomyocytes more efficiently in the presence than in the absence of high glucose. Glucose significantly promoted survival of cardiomyocytes exposed to hypoxia. HMR 1098, an antagonist of sarcolemmal K(ATP) channels, inhibited glucose-induced activation of whole-cell K(+) current during hypoxia as well as glucose-mediated cytoprotection. An inhibitor of glyceraldehyde 3-phosphate dehydrogenase, iodoacetate, inhibited glycolysis in hypoxia and blocked the activation of sarcolemmal K(ATP) channels. Based on the obtained results, we conclude that the activation of sarcolemmal K(ATP) channels is involved in glucose-mediated cardioprotection.     

Quantification of Mg2+ extrusion and cytosolic Mg2+-buffering in Xenopus oocytes

Intracellular Mg(2+) buffering and Mg(2+) extrusion were investigated in Xenopus laevis oocytes. Mg(2+) or EDTA were pressure injected and the resulting changes in the intracellular Mg(2+) concentration were measured simultaneously with Mg(2+)-selective microelectrodes. In the presence of extracellular Na(+), injected Mg(2+) was extruded from the oocytes with an estimated v(max) and K(M) of 74 pmol cm(-2)s(-1) and 1.28 mM, respectively. To investigate genuine cytosolic Mg(2+) buffering, measurements were carried out in the nominal absence of extracellular Na(+) to block Mg(2+) extrusion, and during the application of CCCP (inhibiting mitochondrial uptake). Under these conditions, Mg(2+) buffering calculated after both MgCl(2) and EDTA injections could be described by a buffer equivalent with a concentration of 9.8mM and an apparent dissociation constant, K(d-app), of 0.6mM together with an [ATP](i) of 0.9 mM with a K(d-app) 0.12 mM. Xenopus oocytes thus possess highly efficient mechanisms to maintain their intracellular Mg(2+) concentration.     

Spermine block of the strong inward rectifier potassium channel Kir2.1: dual roles of surface charge screening and pore block

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg(2+). Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.     

Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a role in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.