Features of MotA proton channel structure revealed by tryptophan-scanning mutagenesis.

The MotA protein of Escherichia coli is a component of the flagellar motors that functions in transmembrane proton conduction. Here, we report several features of MotA structure revealed by use of a mutagenesis-based approach. Single tryptophan residues were introduced at many positions within the four hydrophobic segments of MotA, and the effects on function were measured. Function was disrupted according to a periodic pattern that implies that the membrane-spanning segments are alpha-helices and that identifies the lipid-facing parts of each helix. The results support a hypothesis for MotA structure and mechanism in which water molecules form most of the proton-conducting pathway. The success of this approach in studying MotA suggests that it could be useful in structure-function studies of other integral membrane proteins.

Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.

A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus.

Genetic and physiological studies of the Drosophila Hyperkinetic (Hk) mutant revealed defects in the function or regulation of K+ channels encoded by the Shaker (Sh) locus. The Hk polypeptide, determined from analysis of cDNA clones, is a homologue of mammalian K+ channel beta subunits (Kv beta). Coexpression of Hk with Sh in Xenopus oocytes increases current amplitudes and changes the voltage dependence and kinetics of activation and inactivation, consistent with predicted functions of Hk in vivo. Sequence alignments show that Hk, together with mammalian Kv beta, represents an additional branch of the aldo-keto reductase superfamily. These results are relevant to understanding the function and evolutionary origin of Kv beta.

Characterization of a voltage-gated K+ channel beta subunit expressed in human heart.

Voltage-gated K+ channels are important modulators of the cardiac action potential. However, the correlation of endogenous myocyte currents with K+ channels cloned from human heart is complicated by the possibility that heterotetrameric alpha-subunit combinations and function-altering beta subunits exist in native tissue. Therefore, a variety of subunit interactions may generate cardiac K+ channel diversity. We report here the cloning of a voltage-gated K+ channel beta subunit, hKv beta 3, from adult human left ventricle that shows 84% and 74% amino acid sequence identity with the previously cloned rat Kv beta 1 and Kv beta 2 subunits, respectively. Together these three Kv beta subunits share > 82% identity in the carboxyl-terminal 329 aa and show low identity in the amino-terminal 79 aa. RNA analysis indicated that hKv beta 3 message is 2-fold more abundant in human ventricle than in atrium and is expressed in both healthy and diseased human hearts. Coinjection of hKv beta 3 with a human cardiac delayed rectifier, hKv1.5, in Xenopus oocytes increased inactivation, induced an 18-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 8.0 msec vs. 35.4 msec at -50 mV). hKv beta 3 was localized to human chromosome 3 by using a human/rodent cell hybrid mapping panel. These data confirm the presence of functionally important K+ channel beta subunits in human heart and indicate that beta-subunit composition must be accounted for when comparing cloned channels with endogenous cardiac currents.

Elimination of potassium channel expression by antisense oligonucleotides in a pituitary cell line.

The clonal rat pituitary cell line GH4C1 expresses the genes for several voltage-dependent potassium channels including Kv1.5 and Kv1.4. Dexamethasone, a glucocorticoid agonist, induces a slowly inactivating potassium current in these cells but does not alter the amplitude of a rapidly inactivating component of potassium current. We have found that the induction of the slowly inactivating current can be blocked by an antisense phosphorothioate deoxyoligonucleotide to the Kv1.5 mRNA sequence. In contrast, antisense deoxyoligonucleotides against Kv1.4 mRNA specifically decrease the expression of the dexamethasone-insensitive rapidly inactivating current. These results demonstrate the usefulness of antisense oligonucleotides in correlating potassium currents with specific potassium channel proteins in the cell types in which they are naturally expressed.

A corticosteroid-induced gene expressing an "IsK-like" K+ channel activity in Xenopus oocytes.

Screening a rat colon cDNA library for aldosterone-induced genes resulted in the molecular cloning of a cDNA whose corresponding mRNA is strongly induced in the colon by dexamethasone, aldosterone, and a low NaCl diet. A similar mRNA was detected in kidney papilla but not in brain, heart, or skeletal muscle. Xenopus laevis oocytes injected with cRNA synthesized from this clone, designated CHIF (channel-inducing factor), express a K(+)-specific channel activity. The biophysical, pharmacological, and regulatory characteristics of this channel are very similar to those reported before for IsK (minK). These include: slow (tau > 20 s) activation by membrane depolarization with a threshold potential above -50 mV, blockade by clofilium, inhibition by phorbol ester, and activation by 8-bromoadenosine 3',5'-cyclic monophosphate and high cytoplasmic Ca2+. The primary structure of this clone, however, shows no homology to IsK. Instead, CHIF exhibits > 50% similarity to two other short bitopic membrane proteins, phospholemman and the gamma subunit of Na+K(+)-ATPase. The data are consistent with the possibility that CHIF is a member of a family of transmembrane regulators capable of activating endogenous oocyte transport proteins.

A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system.

We have studied the functional consequences of a mutation in the epithelial Na+ channel that causes a heritable form of salt-sensitive hypertension, Liddle disease. This mutation, identified in the original kindred described by Liddle, introduces a premature stop codon in the channel beta subunit, resulting in a deletion of almost all of the C terminus of the encoded protein. Coexpression of the mutant beta subunit with wild-type alpha and gamma subunits in Xenopus laevis oocytes resulted in an approximately 3-fold increase in the macroscopic amiloride-sensitive Na+ current (INa) compared with the wild-type channel. This change in INa reflected an increase in the overall channel activity characterized by a higher number of active channels in membrane patches. The truncation mutation in the beta subunit of epithelial Na+ channel did not alter the biophysical and pharmacological properties of the channel--including unitary conductance, ion selectivity, or sensitivity to amiloride block. These results provide direct physiological evidence that Liddle disease is related to constitutive channel hyperactivity in the cell membrane. Deletions of the C-terminal end of the beta and gamma subunits of rat epithelial Na+ channel were functionally equivalent in increasing INa, suggesting that the cytoplasmic domain of the gamma subunit might be another molecular target for mutations responsible for salt-sensitive forms of hypertension.