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246


Cloning of a human cDNA expressing a high voltage-activating, TEA-sensitive, type-A K+ channel which maps to chromosome 1 band p21

Rudy B; Sen K; Vega-Saenz de Miera E; Lau D; Ried T; Ward DC
Over ten different mammalian genes related to the Drosophila Shaker gene (the Sh gene family) have been identified recently. These genes encode subunits of voltage-dependent K+ channels. The family consists of four subfamilies: ShI genes are homologues of Shaker; ShII, ShIII, and ShIV are homologues of three other Shaker-like genes in Drosophila, Shab, Shaw, and Shal, respectively. We report here the cloning of a human K+ channel ShIII cDNA (HKShIIIC) obtained from a brain stem cDNA library. HKShIIIC transcripts express an atypical voltage-dependent transient (A-type) K+ current in Xenopus oocytes. This current is activated by large membrane depolarizations and is extremely sensitive to the K+ channel blocker TEA unlike most A-type currents. The gene encoding HKShIIIC maps to chromosome 1p21
PMID: 1920536
ISSN: 0360-4012
CID: 13974

Molecular cloning of a member of a third class of Shaker-family K+ channel genes in mammals [Correction]

McCormack T; Vega-Saenz de Miera EC; Rudy B
In the human proteolipid protein gene, the base sequence of the intronic region 5' to exon 6 was found to be 5'-ctctttcattttcctgcag-3' and not 5'-ctctttt-cattttcctgcag-3' as previously reported
PMCID:363768
PMID: 2023956
ISSN: 0027-8424
CID: 18836

A role for hydrophobic residues in the voltage-dependent gating of Shaker K+ channels

McCormack K; Tanouye MA; Iverson LE; Lin JW; Ramaswami M; McCormack T; Campanelli JT; Mathew MK; Rudy B
A leucine heptad repeat is well conserved in voltage-dependent ion channels. Herein we examine the role of the repeat region in Shaker K+ channels through substitution of the leucines in the repeat and through coexpression of normal and truncated products. In contrast to leucine-zipper DNA-binding proteins, we find that the subunit assembly of Shaker does not depend on the leucine heptad repeat. Instead, we report that substitutions of the leucines in the repeat produce large effects on the observed voltage dependence of conductance voltage and prepulse inactivation curves. Our results suggest that the leucines mediate interactions that play an important role in the transduction of charge movement into channel opening and closing
PMCID:51354
PMID: 2011602
ISSN: 0027-8424
CID: 18837

Characterization of maintained voltage-dependent K(+)-channels induced in Xenopus oocytes by rat brain mRNA

Hoger JH; Rudy B; Lester HA; Davidson N
The voltage-dependent K+ currents encoded by rat brain mRNA were studied in Xenopus oocytes after the voltage-dependent Na+ currents and the Ca(2+)-activated Cl- currents were eliminated pharmacologically. This paper describes the maintained K+ currents (IK), defined primarily by resistance to inactivation for 1 s at a holding potential of -40 mV. IK activates at potentials more positive than -60 to -70 mV and consists of both low-threshold and high-threshold components. IK is partially blocked by both tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which appear to be blocking the same component. Long depolarizing pulses result in incomplete inactivation of IK; the inactivating component is inhibited by TEA. Sucrose density gradient fractionation partially resolves the RNA encoding the several components of IK; most IK arises from size classes between 3.8 and 9.5 kb. The study gives further evidence for the existence of numerous distinct RNA populations that encode brain K+ channels different from previously reported cloned K+ channels that have been expressed in Xenopus oocytes
PMID: 1647478
ISSN: 0169-328x
CID: 18838

Families of potassium channel genes in mammals: Toward an understanding of the molecular basis of potassium channel diversity

Rudy, B; Kentros, C; Vela-Saenz De Miera, E
PMID: 19912787
ISSN: 1044-7431
CID: 105342

Human potassium channel genes: Molecular cloning and functional expression

Ramaswami, M; Gautam, M; Kamb, A; Rudy, B; Tanouye, M A; Mathew, M K
Complementary DNAs representing three voltage-gated K(+) channels from humans (HuKI, HuKII, and HuKIV) were isolated, their nucleotide sequences determined, and their functional products examined electrophysiologically. The three human K(+) channels are closely related to the Shaker gene of Drosophila and possess several canonical structural features including multiple hydrophobic segments which are potentially membrane spanning, a positively charged S4 segment which may be the voltage sensor, and a leucine heptad repeat which may be involved in channel gating. Members of the human gene family have specific, highly conserved homologs in rodents, suggesting that the individual members arose prior to the mammalian radiation. The degree of homology indicates that these are among the most highly conserved proteins known. The three human channels expressed in Xenopus oocytes vary in voltage dependence, kinetics, and sensitivity to pharmacological blockers of K(+) channels. HuKII is a rapidly inactivating channel; HuKI and HuKIV are noninactivating. Also, although all three channels are sensitive to the K(+) channel blocker, 4-aminopyridine, only HuKI has tetraethylammonium sensitivity; only HuKIV has charybdotoxin sensitivity. Differences are observed between the pharmacological sensitivities of human channels and the reported sensitivities of their rat homology.
PMID: 19912772
ISSN: 1044-7431
CID: 3689542

Shaker K+ channel subunits from heteromultimeric channels with novel functional properties

McCormack K; Lin JW; Iverson LE; Rudy B
A large number of related genes (the Sh gene family) encode potassium channel subunits which form voltage-dependent K+ channels by aggregating into homomulitimers. One of these genes, the Shaker gene in Drosophila, generates several products by alternative splicing. These products encode proteins with a constant central region flanked by variable amino and carboxyl domains. Coinjection of two Shaker RNAs with different amino or different carboxyl ends into Xenopus oocytes produces K+ currents that display functional properties distinct from those observed when each RNA is injected separately, indicating the formation of heteromultimeric channels. The analysis of Shaker heteromultimers suggests certain rules regarding the roles of variable amino and carboxyl domains in determining kinetic properties of heteromultimeric channels. Heteromultimers with different amino ends produce currents in which the amino end that produces more inactivation dominates the kinetics. In contrast, heteromultimers with different carboxyl ends recover from inactivation at a rate closer to that observed in homomultimers of the subunit which results in faster recovery. While this and other recent reports demonstrate that closely related Sh family proteins form functional heteromultimers, we show here that two less closely related Sh proteins do not seem to form functional heteromultimeric channels. The data suggest that sites for subunit recognition may be found in sequences within a core region, starting about 130 residues before the first membrane spanning domain of Shaker and ending after the last membrane spanning domain, which are not conserved between Sh Class I and Class III genes
PMID: 1699527
ISSN: 0006-291x
CID: 18839

The role of the divergent amino and carboxyl domains on the inactivation properties of potassium channels derived from the Shaker gene of Drosophila

Iverson LE; Rudy B
Several products generated from the Drosophila Shaker gene by alternative splicing predict a group of similar proteins with an identical central and variable amino and carboxyl domains. We have constructed 9 Sh cDNAs combining 3 different 5' domains with 3 different 3' domains. RNA transcribed from 6 of these cDNAs induce K+ currents in Xenopus oocytes. All currents share similar properties of voltage dependence, potassium selectivity, and block by 4-AP, TEA, and charybdotoxin. These properties presumably result from a channel core formed by the identical central region of the proteins. The currents differ in macroscopic inactivation kinetics. Five RNAs induced K+ currents which inactivate, each at distinct rates, during short depolarizations. The sixth RNA induces a current that essentially does not inactivate unless depolarized for many seconds. This raises the possibility that Sh may encode nontransient as well as transient K+ currents. Analysis of currents produced by the various combinations suggests that the divergent amino domains influence the stability of a first, nonabsorbing, inactivated state. This results in striking differences in the probability of channel reopening, as observed in single-channel recordings, of those channels with identical carboxyl but different amino domains. Furthermore, based on macroscopic analysis of the currents, we suggest that the primary role of the carboxyl domains is to influence the relative stability between the first and a second inactivated state. The second inactivated state is essentially absorbing, and recovery from this state is very slow. The observed differences in the rates of recovery from inactivation of channels containing different carboxyl domains reflect differences in the rates at which they enter this second inactivated state
PMID: 1697898
ISSN: 0270-6474
CID: 18840

Differential effects of NGF, FGF, EGF, cAMP, and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells

Pollock JD; Krempin M; Rudy B
PC12 cells are a pheochromocytoma cell line that can be made to differentiate into sympatheticlike neurons by nerve growth factor (NGF). An essential component of the NGF-induced differentiation is the development of action potentials and sodium channels. Using whole-cell clamp we have confirmed that NGF produces a 5- to 6-fold increase in sodium channel density. The sodium channels induced by NGF are not different from those in cells not treated with NGF and are similar to those in other cell types. Basic fibroblast growth factor (FGF), another growth factor that causes PC12 cells to differentiate into sympathetic-like neurons, also produces a 5- to 6-fold increase in sodium current density with channels indistinguishable from those in PC12 cells treated and not treated with NGF. Basic FGF produces the same or somewhat larger increase in sodium channel density but much less neurite outgrowth. In contrast, epidermal growth factor does not produce neurite outgrowth but induces a small, reproducible increase in sodium channel density. Cyclic AMP produces spike-like processes but not neurites and results in a decrease in sodium current and sodium current density. Dexamethasone, a synthetic glucocorticoid, inhibits the increase in sodium current and sodium current density but does not antagonize the neurite outgrowth induced by NGF. Thus, although the increase in sodium channel expression induced by NGF and basic FGF parallels the changes in morphology that lead to neurite outgrowth, it clearly does not depend on them. The results show that different aspects of neuronal differentiation might be independently regulated by the microenvironment
PMID: 2167354
ISSN: 0270-6474
CID: 8471

Molecular cloning of a member of a third class of Shaker-family K+ channel genes in mammals [published erratum appears in Proc Natl Acad Sci U S A 1991 May 1;88(9):4060]

McCormack T; Vega-Saenz de Miera EC; Rudy B
We report the cloning of RKShIIIA, a cDNA encoding a K+ channel sequence expressed in rat brain. This cDNA encodes K+ channel subunits that express in Xenopus oocytes a slow, 4-aminopyridine- and tetraethylammonium-sensitive, delayed rectifier-type K+ channel activated by large membrane depolarizations. This gene belongs to the Shaker (Sh) family of K+ channel genes, since the predicted protein has the same overall structure and shows significant homology to other members of this family. However, RKShIIIA cannot be assigned to either of the two known classes of Sh-family genes in mammals based on sequence analysis. Notable features of the RKShIIIA protein product include a probable cytoplasmic loop rich in prolines and a stretch very homologous to the Drosophila Shaw protein, both near the amino terminus
PMCID:54295
PMID: 2367536
ISSN: 0027-8424
CID: 8472