Faculty Bibliography

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

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

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

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

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.

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

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