Potassium channels control the resting membrane potential and excitability of biological tissues. Many voltage-gated potassium channels are controlled through interactions with accessory subunits of the KCNE family through mechanisms still not known. Gating of mammalian channel KCNQ1 is dramatically regulated by KCNE subunits. We have found that multiple segments of the channel pore structure bind to the accessory protein KCNE1. The sites that confer KCNE1 binding are necessary for the functional interaction, and all sites must be present in the channel together for proper regulation by the accessory subunit. Specific gating control is localized to a single site of interaction between the ion channel and accessory subunit. Thus, direct physical interaction with the ion channel pore is the basis of KCNE1 regulation of K+ channels.

Objective. Nephrogenic systemic fibrosis (NSF) is a rapidly progressive, debilitating condition that causes cutaneous and visceral fibrosis in patients with renal failure. Little is known about its prevalence or etiology. The aim of this study was to establish the prevalence of NSF and associated risk factors Methods. Two cohorts of patients were recruited from 6 outpatient hemodialysis centers and examined for cutaneous changes of NSF, which were defined using a scoring system based on hyperpigmentation, hardening, and tethering of skin on the extremities. Demographic data were gathered, mortality was followed up prospectively for 24 months, and gadolinium exposure was ascertained for a subgroup of patients in the second cohort.Results. Examination reproducibility was 97% in cohort 1. In cohort 2, 25 (13%) of 186 patients demonstrated cutaneous changes of NSF. Twenty-four-month mortality following examination was 48% and 20% in patients with and those without cutaneous changes of NSF, respectively (adjusted hazard ratio 2.9, 95% confidence interval [95% CI] 1.4-5.9). Cutaneous changes of NSF were observed in 16 (30%) of 54 patients with prior exposure to gadopentetate dimeglumine contrast during imaging studies. Exposure to gadoliniumcontaining contrast was associated with an increased risk of developing cutaneous changes of NSF (odds ratio 14.7, 95% CI 1.9-117.0) compared with nonexposed patients.

HERG1 K ؉ channel mutations are responsible for one form of dominantly inherited long QT syndrome (LQT). Some LQT mutations exert a dominant negative effect on wild-type current expression. To investigate mechanisms of dominant-negative behavior, we co-expressed wild-type HERG with the A561V mutant in mammalian cells. Transfection with various cDNA ratios produced HERG K ؉ current densities that approached a predicted binomial distribution where mutant and wild-type subunits co-assemble in a tetramer with nearly complete dominance. Using C terminus myc-tagged wild-type HERG we specifically followed the mutant's effect on full-length wild-type HERG protein expression. Co-expression with A561V reduced the abundance of fulllength wild-type HERG protein comparable to the current reduction. Reduction of wild-type protein was due to decreased synthesis and increased turnover. Conditions facilitating protein folding (growth at 30°C, or in 10% glycerol) resulted in partial rescue from the dominant effect, as did the 26 S proteosome inhibitor ALLN. Thus, for A561V, dominant negative effects result from assembly of wild-type subunits with mutant very early in production leading to rapid recognition of mutant channels and targeting for proteolysis. These results establish protein misfolding, cellular proofreading, and bystander involvement as contributing mechanisms for dominant effects in LQT2. Hereditary long QT syndrome (LQTS)1 is a genetic disorder caused by mutations in one or more of a number of ion channel subunits expressed in the heart (1, 2). Delayed cardiac repolarization, ventricular arrhythmias, syncope, and sudden death characterize LQTS. Although there is a variable spectrum of cardiac electrical abnormalities and penetrance, in the great majority of instances inheritance is dominant. To date, six loci and five genes have been identified as responsible for LQTS (3). One of the involved genes, LQT2, encodes HERG, the poreforming subunit of the rapidly activating delayed rectifier potassium current (I Kr ) (4, 5). Increasing numbers of HERG mutations are being identified through genetic screening of LQTS patients (6 -10). Most of these are missense mutations and are located in membrane spanning regions or the pore domain. Other mutations include an intragenic deletion, a frameshift deletion resulting in a truncated protein, and a splice-acceptor site mutation. Electrophysiological analysis of several of these mutants has shown defects ranging from undetectable current to current with altered gating or ion permeation. In addition, several mutant alleles of HERG dominantly suppress wild-type current (11, 12). Zhou and co-workers (13-15) have shown that abnormal protein trafficking and accelerated degradation of mutant homotetramers contribute to reduced or absent current production in several missense mutations of HERG.In the present study we have investigated mechanisms responsible for the dominant negative behavior of the HERG A561V LQT mutant allele using electrophysiological, biochemical, and pharmacological...

Acute stress provokes lethal cardiac arrhythmias in the hereditary long QT syndrome. Here we provide a novel molecular mechanism linking b-adrenergic signaling and altered human ether-a-go-go related gene (HERG) channel activity. Stress stimulates b-adrenergic receptors, leading to cAMP elevations that can regulate HERG K + channels both directly and via phosphorylation by cAMP-dependent protein kinase (PKA). We show that HERG associates with 14-3-3e to potentiate cAMP/PKA effects upon HERG. The binding of 14-3-3 occurs simultaneously at the N-and C-termini of the HERG channel. 14-3-3 accelerates and enhances HERG activation, an effect that requires PKA phosphorylation of HERG and dimerization of 14-3-3. The interaction also stabilizes the lifetime of the PKA-phosphorylated state of the channel by shielding the phosphates from cellular phosphatases. The net result is a prolongation of the effect of adrenergic stimulation upon HERG activity. Thus, 14-3-3 interactions with HERG may provide a unique mechanism for plasticity in the control of membrane excitability and cardiac rhythm.

Cardiac IKs, the slowly activated delayed-rectifier K+ current, is produced by the protein complex composed of α- and β-subunits: KvLQT1 and minK. Mutations of genes encoding KvLQT1 and minK are responsible for the hereditary long QT syndrome (loci LQT1 and LQT5, respectively). MinK-L51H fails to traffic to the cell surface, thereby failing to produce effective IKs. We examined the effects that minK-L51H and an endoplasmic reticulum (ER)-targeted minK (minK-ER) exerted over the electrophysiology and biosynthesis of coexpressed KvLQT1. Both minK-L51H and minK-ER were sequestered primarily in the ER as confirmed by lack of plasma membrane expression. Glycosylation and immunofluorescence patterns of minK-L51H were qualitatively different for minK-ER, suggesting differences in trafficking. Cotransfection with the minK mutants resulted in reduced surface expression of KvLQT1 as assayed by whole cell voltage clamp and immunofluorescence. MinK-L51H reduced current amplitude by 91% compared with wild-type (WT) minK/KvLQT1, and the residual current was identical to KvLQT1 without minK. The phenotype of minK-L51H on IKs was not dominant because coexpressed WT minK rescued the current and surface expression. Collectively, our data suggest that ER quality control prevents minK-L51H/KvLQT1 complexes from trafficking to the plasma membrane, resulting in decreased IKs. This is the first demonstration that a minK LQT mutation is capable of conferring trafficking defects onto its associated α-subunit.

The sol-gel encapsulation process has been exploited in recent years for the immobilization of proteins to be used as biosensors. Sol-gels derived from tetramethyl orthosilicate provide a stable environment for the macromolecule combined with the free flow of small substrates to a protein's binding site. The functionality of a number of enzymes within the solid matrix has been demonstrated. However, very little biophysical characterization of the encapsulated proteins has been done. In this study, time-resolved fluorescence anisotropy was used to compare the rotational mobility of two probes in sol-gel matrices derived from three different preparative methods. A small fluorescent probe, sulforhodamine 101 (SR101), was used to gauge the relative solvent viscosity within the sol-gels. Magnesium protoporphyrin IX-substituted myoglobin (MgMb) provides a convenient fluorescent probe for measuring rotational dynamics of a typical globular protein. The anisotropy decay of the Mg-heme is sensitive only to the global protein motion. The SR101 reveals both low ( < 1 ns) and high ( ) 6-500 ns) viscosity encapsulation sites within the matrix, and the populations of these sites are dependent on gel preparation and age. The protein, however, shows greatly diminished decay of the fluorescence anisotropy ( ∼ 1 µs) in two of the three gels (but was denatured in the third). This is consistent with restrictive encapsulation sites where size and/or environment substantially impedes rotational diffusion.

No abstract

We previously reported that cloned human ether-à-go-go-related gene (HERG) K ϩ channels are regulated by changes in phosphatidylinositol 4,5-bisphosphate (PIP2) concentration. Here we investigated the molecular determinants of PIP2 interactions with HERG channel protein. To establish the molecular nature of the PIP 2-HERG interaction, we examined a segment of the HERG COOH terminus with a high concentration of positively charged amino acids (nos. 883-894) as a possible site of interaction with negatively charged PIP 2. When we excised deletion-HERG (D-HERG) or mutated methionine-substituted-HERG (M-HERG) this segment of HERG to neutralize the amino acid charge, the mutant channels produced current that was indistinguishable from wild-type HERG. Elevating internal PIP 2, however, no longer accelerated the activation kinetics of the mutant HERG. Moreover, PIP2-dependent hyperpolarizing shifts in the voltage dependence of activation were abolished with both mutants. PIP2 effects on channelinactivation kinetics remained intact, which suggests an uncoupling of inactivation and activation regulation by PIP 2. The specific binding of radiolabeled PIP2 to both mutant channel proteins was nearly abolished. Stimulation of ␣1A-adrenergic receptors produced a reduction in current amplitude of the rapidly activating delayed rectifier K ϩ current (the current carried by ERG protein) from rabbit ventricular myocytes. The ␣-adrenergic-induced current reduction was accentuated by PKC blockers and also unmasked a depolarizing shift in the voltage dependence of activation, which supports the conclusion that receptor activation of PLC results in PIP 2 consumption that alters channel activity. These results support a physiological role for PIP2 regulation of the rapidly activating delayed rectifier K ϩ current during autonomic stimulation and localize a site of interaction to the COOHterminal tail of the HERG K ϩ channel.human ether-à-go-go-related gene; phosphatidylinositol 4,5-bisphosphate; delayed rectifier K ϩ current; channel; phospholipids; G protein-coupled receptor; mutagenesis; phospholipase C REGULATION OF ION CHANNEL function plays a pivotal role in the control of heart rate and contractility via changes in cardiac myocyte excitability. Humoral mediators and receptors are involved in determining channel responses to changing cardiovascular demands. The dynamic beat-to-beat regulation of ion channels is precisely controlled by autonomic stimulation through complex interplay of second messengers, kinases, G proteins, and protein-protein interactions. The rapidly activating delayed rectifier K ϩ current (I Kr ) is essential for proper repolarization of the cardiac myocyte at the end of each action potential (15, 23). Decreased abundance or malfunction of I Kr increases the propensity to ventricular tachyarrhythmia (32). The gene that encodes the pore-forming subunit of the I Kr channel is human ether-à-go-go-related gene (HERG), which has been linked to both hereditary and acquired ventricular arrhythmias (8,19,22,29). Adren...