Structure of thiocyanate hydrolase: a new nitrile hydratase family protein with a novel five-coordinate cobalt(III) center

Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt(III)-containing enzyme catalyzing the degradation of thiocyanate to carbonyl sulfide and ammonia. We determined the crystal structures of the apo- and native SCNases at a resolution of 2.0 A. SCNases in both forms had a conserved hetero-dodecameric structure, (alphabetagamma)(4). Four alphabetagamma hetero-trimers were structurally equivalent. One alphabetagamma hetero-trimer was composed of the core domain and the betaN domain, which was located at the center of the molecule and linked the hetero-trimers with novel quaternary interfaces. In both the apo- and native SCNases, the core domain was structurally conserved between those of iron and cobalt-types of nitrile hydratase (NHase). Native SCNase possessed the post-translationally modified cysteine ligands, gammaCys131-SO(2)H and gammaCys133-SOH like NHases. However, the low-spin cobalt(III) was found to be in the distorted square-pyramidal geometry, which had not been reported before in any protein. The size as well as the electrostatic properties of the substrate-binding pocket was totally different from NHases with respect to the charge distribution and the substrate accessibility, which rationally explains the differences in the substrate preference between SCNase and NHase.     

Post-translational modification is essential for catalytic activity of nitrile hydratase

Nitrile hydratase from Rhodococcus sp. N-771 is an alphabeta heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, alphaCys112 and alphaCys114 are modified to a cysteine sulfinic acid (Cys-SO2H) and a cysteine sulfenic acid (Cys-SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The alphabeta complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC/MS analysis showed that the anaerobically reconstituted alphabeta complex did not have the modification of alphaCys112-SO2H and aerobic incubation induced the modification. The activity of the reconstituted alphabeta complex correlated with the amount of alphaCys112-SO2H. Furthermore, ESI-LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that alphaCys114 is modified to Cys-SOH together with the sulfinic acid modification of alphaCys112. These results suggest that alphaCys112 and alphaCys114 are spontaneously oxidized to Cys-SO2H and Cys-SOH, respectively, and alphaCys112-SO2H is responsible for the catalytic activity solely or in combination with alphaCys114-SOH.     

Subunit fusion of two yeast d-amino acid oxidases enhances their thermostability and resistance to H2O2

D-amino acid oxidases from Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) are both yeast homodimeric flavoenzymes. Two of their cDNA genes were connected by a hexanucleotide linker and heterologously expressed in E. coli to produce the corresponding double DAOs (dRtDAO and dTvDAO) with two subunits fused into a single polypeptide. The specific activities of double DAOs remained similar to those of native dimeric DAOs, although the catalytic efficiencies (k(cat)/K(M)) were decreased due to higher K(M) values. The T(m) value for dRtDAO was shifted 5 degrees C higher while that for dTvDAO was increased only by 2 degrees C, in comparison with the corresponding native counterparts. In the presence of 10 mM H(2)O(2), dRtDAO and dTvDAO exhibited half-lives of about 60 and 40 min, respectively, which were 2- and 1.5-fold, respectively, longer than their native DAOs. These yeast DAOs can therefore be thermally and oxidatively stabilized by linking their subunits together.     

Peptide inhibition of constitutively activated epithelial Na(+) channels expressed in Xenopus oocytes

The hypothesis that 30-amino acid peptides corresponding to the C-terminal portion of the beta- and/or gamma-rat epithelial sodium channel (rENaC) subunits block constitutively activated ENaC was tested by examining the effects of these peptides on wild-type (wt) rENaC (alphabetagamma-rENaC), truncated Liddle's mutants (alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC), and point mutants (alphabeta(Y)gamma-, alphabetagamma(Y)-rENaC) expressed in Xenopus oocytes. The chord conductances of alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC were 2- or 3-fold greater than for wt alphabetagamma-rENaC. Introduction of peptides into oocytes expressing alphabeta(T)gamma-, alphabetagamma(T)-, and alphabeta(T)gamma(T)-rENaC produced a concentration-dependent inhibition of the amiloride-sensitive Na(+) conductances, with apparent dissociation constants (K(d)) ranging from 1700 to 160 microM, depending upon whether individual peptides or their combination was used. Injection of peptides alone or in combination into oocytes expressing wt alphabetagamma-rENaC or single-point mutants did not affect the amiloride-sensitive whole-cell currents. The single channel conductances of all the mutant ENaCs were the same as that of wild type (alphabetagamma-). The single channel activities (N.P(o)) of the mutants were approximately 2.2-2.6-fold greater than wt alphabetagamma-rENaC (1.08 +/- 0.24, n = 7) and were reduced to 1.09 +/- 0.17 by 100 microM peptide mixture (n = 9). The peptides were without effect on the single channel properties of either wt or single-point mutants of rENaC. Our data demonstrate that the C-terminal peptides blocked the Liddle's truncation mutant (alphabeta(T)gamma(T)) expressed in Xenopus oocytes but not the single-point mutants (alphabeta(Y)gamma or alphabetagamma(Y)). Moreover, the blocking effect of both peptides in combination on alphabeta(T)gamma(T)-rENaC was synergistic.     

Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage

Chondroitin sulfate E (CS-E), a chondroitin sulfate isomer containing GlcAbeta1-3GalNAc(4,6-SO(4)) repeating unit, was found in various mammalian cells in addition to squid cartilage and is predicted to have several physiological functions in various mammalian systems such as mast cell maturation, regulation of procoagulant activity of monocytes, and binding to midkine or chemokines. To clarify the physiological functions of GalNAc(4,6-SO(4)) repeating unit, preparation of CS-E with a defined content of GalNAc(4,6-SO(4)) residues is important. We report here the in vitro synthesis of CS-E from chondrotin sulfate A (CS-A) by the purified squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) which catalyzed transfer of sulfate from 3(')-phosphoadenosine-5(')-phosphosulfate to position 6 of GalNAc(4SO(4)) residues of CS-A and dermatan sulfate (DS). When CS-A was used as an acceptor, about half of GalNAc(4SO(4)) residues, on average, were converted to GalNAc(4,6-SO(4)) residues. Anion exchange chromatography of the CS-E synthesized in vitro showed marked heterogeneity in negative charge; the proportion of GalNAc(4,6-SO(4)) in the most negative fraction exceeded 70% of the total sulfated repeating units. GalNAc4S-6ST also catalyzed the synthesis of oversulfated DS with GalNAc(4,6-SO(4)) residues from DS. Squid GalNAc4S-6ST thus should provide a useful tool for preparing CS-E and oversulfated DS with a defined proportion of GalNAc(4,6-SO(4)) residues.     

Synergistic activation of G protein-gated inwardly rectifying potassium channels by the betagamma subunits of G proteins and Na(+) and Mg(2+) ions.

Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the betagamma subunits of GTP-binding (G) proteins. The presence of phosphatidylinositol-bis-phosphate (PIP(2)) is required for G protein activation. Formation (via hydrolysis of ATP) of endogenous PIP(2) or application of exogenous PIP(2) increases the mean open time of GIRK channels and sensitizes them to gating by internal Na(+) ions. In the present study, we show that the activity of ATP- or PIP(2)-modified channels could also be stimulated by intracellular Mg(2+) ions. In addition, Mg(2+) ions reduced the single-channel conductance of GIRK channels, independently of their gating ability. Both Na(+) and Mg(2+) ions exert their gating effects independently of each other or of the activation by the G(betagamma) subunits. At high levels of PIP(2), synergistic interactions among Na(+), Mg(2+), and G(betagamma) subunits resulted in severalfold stimulated levels of channel activity. Changes in ionic concentrations and/or G protein subunits in the local environment of these K(+) channels could provide a rapid amplification mechanism for generation of graded activity, thereby adjusting the level of excitability of the cells.     

Crystal structure of the ternary FimC-FimF(t)-FimD(N) complex indicates conserved pilus chaperone-subunit complex recognition by the usher FimD

Type 1 pili, anchored to the outer membrane protein FimD, enable uropathogenic Escherichia coli to attach to host cells. During pilus biogenesis, the N-terminal periplasmic domain of FimD (FimD(N)) binds complexes between the chaperone FimC and pilus subunits via its partly disordered N-terminal segment, as recently shown for the FimC-FimH(P)-FimD(N) ternary complex. We report the structure of a new ternary complex (FimC-FimF(t)-FimD(N)) with the subunit FimF(t) instead of FimH(p). FimD(N) recognizes FimC-FimF(t) and FimC-FimH(P) very similarly, predominantly through hydrophobic interactions. The conserved binding mode at a "hot spot" on the chaperone surface could guide the design of pilus assembly inhibitors.     

Protein kinase A is central for forward transport of two-pore domain potassium channels K2P3.1 and K2P9.1

Acid-sensitive two-pore domain potassium channels (K2P3.1 and K2P9.1) play key roles in both physiological and pathophysiological mechanisms, the most fundamental of which is control of resting membrane potential of cells in which they are expressed. These background "leak" channels are constitutively active once expressed at the plasma membrane, and hence tight control of their targeting and surface expression is fundamental to the regulation of K(+) flux and cell excitability. The chaperone protein, 14-3-3, binds to a critical phosphorylated serine in the channel C termini of K2P3.1 and K2P9.1 (Ser(393) and Ser(373), respectively) and overcomes retention in the endoplasmic reticulum by βCOP. We sought to identify the kinase responsible for phosphorylation of the terminal serine in human and rat variants of K2P3.1 and K2P9.1. Adopting a bioinformatic approach, three candidate protein kinases were identified: cAMP-dependent protein kinase, ribosomal S6 kinase, and protein kinase C. In vitro phosphorylation assays were utilized to determine the ability of the candidate kinases to phosphorylate the channel C termini. Electrophysiological measurements of human K2P3.1 transiently expressed in HEK293 cells and cell surface assays of GFP-tagged K2P3.1 and K2P9.1 enabled the determination of the functional implications of phosphorylation by specific kinases. All of our findings support the conclusion that cAMP-dependent protein kinase is responsible for the phosphorylation of the terminal serine in both K2P3.1 and K2P9.1.     

Metal specificity is correlated with two crucial active site residues in Escherichia coli alkaline phosphatase

Escherichia coli alkaline phosphatase exhibits maximal activity when Zn(2+) fills the M1 and M2 metal sites and Mg(2+) fills the M3 metal site. When other metals replace the zinc and magnesium, the catalytic efficiency is reduced by more than 5000-fold. Alkaline phosphatases from organisms such as Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity and function poorly with zinc and magnesium. Previous studies have shown that the D153H alkaline phosphatase exhibited very little activity in the presence of cobalt, while the K328W and especially the D153H/K328W mutant enzymes can use cobalt for catalysis. To understand the structural basis for the altered metal specificity and the ability of the D153H/K328W enzyme to utilize cobalt for catalysis, we determined the structures of the inactive wild-type E. coli enzyme with cobalt (WT_Co) and the structure of the active D153H/K328W enzyme with cobalt (HW_Co). The structural data reveal differences in the metal coordination and in the strength of the interaction with the product phosphate (P(i)). Since release of P(i) is the slow step in the mechanism at alkaline pH, the enhanced binding of P(i) in the WT_Co structure explains the observed decrease in activity, while the weakened binding of P(i) in the HW_Co structure explains the observed increase in activity. These alterations in P(i) affinity are directly related to alterations in the coordination of the metals in the active site of the enzyme.     

Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric alphabetagamma complexes. The alpha and gamma (not beta) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (alpha, beta, gamma, alphabeta, alphagamma, betagamma, and alphabetagamma). We assayed corresponding channel function as amiloride-sensitive sodium transport (I(Na)). We varied furin-mediated proteolysis by mutating the P1 site in alpha and/or gamma subunit furin consensus cleavage sites (alpha(mut) and gamma(mut)). Our findings were as follows. (i) The beta subunit alone is not transported to the cell surface nor cleaved upon assembly with the alpha and/or gamma subunits. (ii) The alpha subunit alone (or in combination with beta and/or gamma) is efficiently transported to the cell surface; a surface-expressed 65-kDa alpha ENaC fragment is undetected in alpha(mut)betagamma, and I(Na) is decreased by 60%. (iii) The gamma subunit alone does not appear at the cell surface; gamma co-expressed with alpha reaches the surface but is not detectably cleaved; and gamma in alphabetagamma complexes appears mainly as a 76-kDa species in the surface pool. Although basal I(Na) of alphabetagamma(mut) was similar to alphabetagamma, gamma(mut) was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of alpha and gamma in alphabetagamma heteromers. Basal I(Na) is reduced by preventing furin-mediated cleavage of the alpha, but not gamma, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.     

ATP-sensitive K+-channel subunits on the mitochondria and endoplasmic reticulum of rat cardiomyocytes.

ATP-sensitive K(+) (K(ATP)) channel subunits on the subcellular structures of rat cardiomyocytes were studied with antibodies against Kir6.1 and Kir6.2. According to the results of Western blot analysis, Kir6.1 was strongly expressed in mitochondrial and microsome fractions, and faintly expressed in cell membrane fraction, whereas Kir6.2 was mainly expressed in the microsome fraction and weakly in cell membrane and mitochondrial fractions. Immunohistochemistry showed that Kir6.1 and Kir6.2 were expressed in the endocardium, atrial and ventricular myocardium, and in vascular smooth muscles. Immunoelectron microscopy revealed that Kir6.1 immunoreactivity was mainly localized in the mitochondria, whereas Kir6.2 immunoreactivity was mainly localized in the endoplasmic reticulum and a few in the mitochondria. Both Kir6.1 and Kir6.2 are candidates of mitochondrial K(ATP) channel subunits. The data obtained in this study will be useful for analyzing the composition of K(ATP) channels of cardiomyocytes and help to understanding the cardioprotective role of K(ATP) channels during heart ischemia.     

Expression of water-soluble, ligand-binding concatameric extracellular domains of the human neuronal nicotinic receptor alpha4 and beta2 subunits in the yeast Pichia pastoris: glycosylation is not required for ligand binding

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that are responsible for cell communication via the neurotransmitter acetylcholine. The predominant nAChR subtype in the mammalian brain with a high affinity for nicotine is composed of α4 and β2 subunits. This nAChR subtype is responsible for addiction to nicotine and is thought to be implicated in Alzheimer and Parkinson diseases and therefore presents an important target for drug design. In an effort to obtain water-soluble, ligand-binding domains of the human α4β2 nAChR for structural studies, we expressed the extracellular domains (ECDs) of these subunits in the eukaryotic expression system Pichia pastoris. The wild-type ECDs and their mutants containing the more hydrophilic Cys-loop from the snail acetylcholine-binding protein (individually expressed or coexpressed) did not demonstrate any specific interaction with ligands. We then linked the mutated ECDs with the 24-amino acid peptide (AGS)(8) and observed that the β2-24-α4 ECD concatamer, but not the α4-24-β2 one, exhibited very satisfactory water solubility and ligand binding properties. The (125)I-epibatidine and [(3)H]nicotine bound to β2-24-α4 with dissociation constants (K(d)) of 0.38 and 19 nm, respectively, close to the published values for the intact α4β2 AChR. In addition, (125)I-epibatidine binding was blocked by nicotine, cytisine, acetylcholine, and carbamylcholine with inhibition constants (K(i)) of 20.64, 3.24, 242, and 2,254 nm, respectively. Interestingly, deglycosylation of the concatamer did not affect its ligand binding properties. Furthermore, the deglycosylated β2-24-α4 ECD existed mainly in monomeric form, thus forming an appropriate material for structural studies and possibly for pharmacological evaluation of novel α4β2 nAChR-specific agonists.     

The crystal structure of calcium- and integrin-binding protein 1: insights into redox regulated functions

Calcium- and integrin-binding protein 1 (CIB1) is involved in the process of platelet aggregation by binding the cytoplasmic tail of the alpha(IIb) subunit of the platelet-specific integrin alpha(Iib)beta(3). Although poorly understood, it is widely believed that CIB1 acts as a global signaling regulator because it is expressed in many tissues that do not express integrin alpha(Iib)beta(3). We report the structure of human CIB1 to a resolution of 2.3 A, crystallized as a dimer. The dimer interface includes an extensive hydrophobic patch in a crystal form with 80% solvent content. Although the dimer form of CIB1 may not be physiologically relevant, this intersub-unit surface is likely to be linked to alpha(IIb) binding and to the binding of other signaling partner proteins. The C-terminal domain of CIB1 is structurally similar to other EF-hand proteins such as calmodulin and calcineurin B. Despite structural homology to the C-terminal domain, the N-terminal domain of CIB1 lacks calcium-binding sites. The structure of CIB1 revealed a complex with a molecule of glutathione in the reduced state bond to the N-terminal domain of one of the two subunits poised to interact with the free thiol of C35. Glutathione bound in this fashion suggests CIB1 may be redox regulated. Next to the bound GSH, the orientation of residues C35, H31, and S48 is suggestive of a cysteine-type protein phosphatase active site. The potential enzymatic activity of CIB1 is discussed and suggests a mechanism by which it regulates a wide variety of proteins in cells in addition to platelets.     

N-terminal deletion of the gamma subunit affects the stabilization and activity of chloroplast ATP synthase

Five truncation mutants of chloroplast ATP synthase gamma subunit from spinach (Spinacia oleracea) lacking 8, 12, 16, 20 or 60 N-terminal amino acids were generated by PCR by a mutagenesis method. The recombinant gamma genes were overexpressed in Escherichia coli and assembled with alphabeta subunits into a native complex. The wild-type (WT) alphabetagamma assembly i.e. alphabetagammaWT exhibited high (Mg2+)-dependent and (Ca2+)-dependent ATP hydrolytic activity. Deletions of eight residues of the gamma subunit N-terminus caused a decrease in rates of ATP hydrolysis to 30% of that of the alphabetaWT assembly. Furthermore, only approximately 6% of ATP hydrolytic activity was retained with the sequential deletions of gamma subunit up to 20 residues compared with the activity of the alphabetaWT assembly. The inhibitory effect of the epsilon subunit on ATP hydrolysis of these alphabetagamma assemblies varied to a large extent. These observations indicate that the N-terminus of the gamma subunit is very important, together with other regions of the gamma subunit, in stabilization of the enzyme complex or during cooperative catalysis. In addition, the in vitro binding assay showed that the gamma subunit N-terminus is not a crucial region in binding of the epsilon subunit.     

Interaction of nucleoredoxin with protein phosphatase 2A

A trimeric protein phosphatase 2A (PP2A(T55)) composed of the catalytic (PP2Ac), structural (PR65/A), and regulatory (PR55/B) subunits was isolated from rabbit skeletal muscle by thiophosphorylase affinity chromatography, and contained two additional proteins of 54 and 55 kDa, respectively. The 54 kDa protein was identified as eukaryotic translation termination factor 1 (eRF1) and as a PP2A interacting protein. The 55 kDa protein is now identified as nucleoredoxin (NRX). The formation of a complex between GST-NRX, PP2A(C) and PP2A(D) was demonstrated by pull-down experiments with purified forms of PP2A, and by immunoprecipitation of HA-tagged NRX expressed in HEK293 cells complexed endogenous PP2A subunits. Analysis of PP2A activity in the presence of GST-NRX showed that NRX competed with polycations for both stimulatory and inhibitory effects on different forms of PP2A.     

Identification of the two histidine residues responsible for the inhibition by malonyl-CoA in peroxisomal carnitine octanoyltransferase from rat liver

Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA. cDNAs encoding full-length wild-type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae. Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity. The wild-type-expressed COT was inhibited by malonyl-CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2-200 microM). These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA. Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.     

The N-terminal regions of beta and gamma subunits lower the solubility of adenosylcobalamin-dependent diol dehydratase

Adenosylcobalamin-dependent diol dehydratase is one of essential components of carboxysome-like polyhedral bodies. It exists as a heterohexamer (alphabetagamma)(2), and its activity is recovered in a precipitant fraction of Klebsiella oxytoca and overexpressing Escherichia coli cells. Limited proteolysis of the enzyme with trypsin converted the enzyme into a highly soluble form without loss of enzyme activity. The N-terminal amino acid sequencing of the enzyme thus solubilized indicated that the N-terminal 20 and 16 amino acid residues had been removed from the beta and gamma subunits, respectively. Mutant enzymes with the same N-terminal truncations of either or both of the beta and gamma subunits were expressed on a high level in E. coli cells. All the mutant enzymes obtained were expressed in a soluble, active form. These results indicate that the N-terminal regions of the beta and gamma subunits lower the solubility of diol dehydratase. The mutant enzyme with the N-terminal truncations of both beta and gamma subunits was essentially indistinguishable in catalytic properties from recombinant wild-type enzyme or the enzyme purified from K. oxytoca in a soluble form.     

Hydroxyzine, promethazine and thioridazine interaction with phospholipid monomolecular layers at the air-water interface

In this work the interaction of Hydroxyzine, Promethazine and Thioridazine with Langmuir films of dipalmitoylphosphatidylcholine (dpPC) and dipalmitoylphosphatidic acid (dpPA), is studied. Temporal variations in lateral surface pressure (pi) were measured at different initial pi (pi(i)), subphase pH and drug-concentration. Drugs with the smallest (PRO) and largest (HYD) molecular size exhibited the lowest adsorption (k(a)) and the highest desorption (k(d)) rate constant values, respectively. The affinity binding constants (K(b)) obtained in monolayers followed the same profile (K(b,PRO) < K(b,HYD) < K(b,THI)) of the egg-PC/water partition coefficients (P) determined in bilayers. The drug concentration required to reach the half-maximal Deltapi at pi(i) = 14 mN/m (K(0.5)), was very sensitive to pH. The maximal increment in pi upon drug incorporation into the monolayer (deltapi(max)) will depend on the phospholipid collapse pressure (pi(c)), the monolayers's compressibility and drug's size, shape and charge. The higher pi(c) of dpPC lead to higher pi(cut-off) values (maximal pi allowing drug penetration), if compared with dpPA. In dpPC and dpPA pi(cut-off) decreased as a function of the molecular size of the uncharged drugs. In dpPA, protonated drugs became electrostatically trapped at the monolayer surface hence drug penetration, monolayer deformation and pi increase were impaired and the correlation between pi(cut-off) and drug molecular size was lost.     

Altered state dependence of c-type inactivation in the long and short forms of human Kv1.5

Evidence from both human and murine cardiomyocytes suggests that truncated isoforms of Kv1.5 can be expressed in vivo. Using whole-cell patch-clamp recordings, we have characterized the activation and inactivation properties of Kv1.5DeltaN209, a naturally occurring short form of human Kv1.5 that lacks roughly 75% of the T1 domain. When expressed in HEK 293 cells, this truncated channel exhibited a V(1/2) of -19.5 +/- 0.9 mV for activation and -35.7 +/- 0.7 mV for inactivation, compared with a V(1/2) of -11.2 +/- 0.3 mV for activation and -0.9 +/- 1.6 mV for inactivation in full-length Kv.15. Kv1.5DeltaN209 channels exhibited several features rarely observed in voltage-gated K(+) channels and absent in full-length Kv1.5, including a U-shaped voltage dependence of inactivation and "excessive cumulative inactivation," in which a train of repetitive depolarizations resulted in greater inactivation than a continuous pulse. Kv1.5DeltaN209 also exhibited a stronger voltage dependence to recovery from inactivation, with the time to half-recovery changing e-fold over 30 mV compared with 66 mV in full-length Kv1.5. During trains of human action potential voltage clamps, Kv1.5DeltaN209 showed 30-35% greater accumulated inactivation than full-length Kv1.5. These results can be explained with a model based on an allosteric model of inactivation in Kv2.1 (Klemic, K.G., C.-C. Shieh, G.E. Kirsch, and S.W. Jones. 1998. Biophys. J. 74:1779-1789) in which an absence of the NH(2) terminus results in accelerated inactivation from closed states relative to full-length Kv1.5. We suggest that differential expression of isoforms of Kv1.5 may contribute to K(+) current diversity in human heart and many other tissues.