Architecture of a K+ channel inner pore revealed by stoichiometric covalent modification.
(25/2415)
Inwardly rectifying K+ channels bind intracellular magnesium and polyamines to generate inward rectification. We have examined the architecture of the inner pore of Kir2.1 channels by covalently attaching a constrained number (from one to four) of positively charged moieties of different sizes to the channel. Our results indicate that the inner pore is formed solely by the second transmembrane segment and is unprecedentedly wide. At a position critical for inward rectification (D172), the pore is sufficiently wide to bind three Mg2+ ions or polyamine molecules simultaneously. Single-channel recordings directly demonstrate that partially modified channels exhibit distinct subconductance levels. Such a wide inner pore may greatly facilitate ion permeation and high-affinity binding of multiple pore blockers to generate strong inward rectification. (+info)
Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors.
(26/2415)
To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell's exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for beta-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 x 10(7) pfu/ml were sufficient for expression in 50% of the approximately 1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel-GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by -17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function. (+info)
Effects of intra- and extracellular acidifications on single channel Kir2.3 currents.
(27/2415)
1. The inward rectifier K+ channel Kir2.3 is inhibited by hypercapnia, and this inhibition may be mediated by decreases in intra- and extracellular pH. To understand whether Kir2.3 has two distinct pH sensors and whether cytosol-soluble factors are involved in the modulation of this channel during intracellular acidification, single channel currents were studied by expressing Kir2.3 in Xenopus oocytes. 2. In excised inside-out patches, Kir2.3 currents had a high baseline channel open-state probability (Po, at pH 7.4) with a strong inward rectification. Single channel conductance at hyperpolarizing membrane potential was about 17 pS with 150 mM K+ applied to both sides of the membrane. The channel showed a substate conductance of about 8 pS. 3. Reduction of intracellular pH (pHi) produced a fast and reversible inhibition of single channel Kir2.3 currents in inside-out patches. The extent of this inhibition is concentration dependent. A clear reduction in Kir2.3 currents was seen at pHi 7.0, and channel activity was completely suppressed at pHi 6.2 with mid-point inhibition (pK) at pH 6.77. 4. The effect of low pHi on Kir2.3 currents was due to a strong inhibition of Po and a moderate suppression of single channel conductance. The pK values for these single channel properties were pH 6.78 and 6.67, respectively. 5. The decrease in Po with low pHi resulted from an increase in the channel mean closed time without significant changes in the mean open time. Substate conductance was not seen during low pHi. 6. Decrease in extracellular pH (pHo) also caused inhibition of single channel activity of Kir2.3 currents in excised outside-out patches. This effect, however, was clearly different from that of pHi: the pK (pH 6.70) was about 0.1 pH units lower; more than 50 % channel activity was retained at pHo 5.8; and low pHo affected mainly single channel conductance. 7. These results therefore indicate that (1) there are two distinct pH sensors in Kir2.3, (2) different mechanisms are involved in the modulation of Kir2.3 through these two pH sensors, and (3) cytosol-soluble factors do not appear to be engaged in this modulation. (+info)
Clinical features of 52 neonates with hyperinsulinism.
(28/2415)
BACKGROUND: Neonatal hyperinsulinemic hypoglycemia is often resistant to medical therapy and is often treated with near-total pancreatectomy. However, the pancreatic lesions may be focal and treatable by partial pancreatic resection. METHODS: We studied 52 neonates with hyperinsulinism who were treated surgically. The type and location of the pancreatic lesions were determined by preoperative pancreatic catheterization and intraoperative histologic studies. Partial pancreatectomy was performed in infants with focal lesions, and near-total pancreatectomy was performed in those with diffuse lesions. The postoperative outcome was determined by measurements of plasma glucose and glycosylated hemoglobin and by oral glucose-tolerance tests. RESULTS: Thirty neonates had diffuse beta-cell hyperfunction, and 22 had focal adenomatous islet-cell hyperplasia. Among the latter, the lesions were in the head of the pancreas in nine, the isthmus in three, the body in eight, and the tail in two. The clinical manifestations were similar in both groups. The infants with focal lesions had no symptoms of hypoglycemia and had normal preprandial and postprandial plasma glucose and glycosylated hemoglobin values and normal results on oral glucose-tolerance tests after partial pancreatectomy (performed in 19 of 22 neonates). By contrast, after near-total pancreatectomy, 13 of the patients with diffuse lesions had persistent hypoglycemia, type 1 diabetes mellitus developed in 8, and hyperglycemia developed in another 7; overall, only 2 patients with diffuse lesions had normal plasma glucose concentrations in the first year after surgery. CONCLUSIONS: Among neonates with hyperinsulinism, about half may have focal islet-cell hyperplasia that can be treated with partial pancreatectomy. These neonates can be identified through pancreatic catheterization and intraoperative histologic studies. (+info)
Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation.
(29/2415)
The rapid atrial rate during atrial fibrillation (AF) decreases the ionic current density of transient outward K+ current, L-type Ca2+ current, and Na+ current, thereby altering cardiac electrophysiology and promoting arrhythmia maintenance. To assess possible underlying changes in cardiac gene expression, we applied competitive reverse transcriptase-polymerase chain reaction to quantify mRNA concentrations in dogs subjected to 7 (group P7 dogs) or 42 (group P42 dogs) days of atrial pacing at 400 bpm and in sham controls. Rapid pacing reduced mRNA concentrations of Kv4.3 (putative gene encoding transient outward K+ current; by 60% in P7 and 74% in P42 dogs; P<0.01 and P<0.001, respectively, versus shams), the alpha1c subunit of L-type Ca2+ channels (by 57% in P7 and 72% in P42 dogs; P<0.01 versus shams for each) and the alpha subunit of cardiac Na+ channels (by 18% in P7 and 42% in P42; P=NS and P<0.01, respectively, versus shams) genes. The observed changes in ion channel mRNA concentrations paralleled previously measured changes in corresponding atrial ionic current densities. Atrial tachycardia did not affect mRNA concentrations of genes encoding delayed or Kir2.1 inward rectifier K+ currents (of which the densities are unchanged by atrial tachycardia) or of the Na+,Ca2+ exchanger. Western blot techniques were used to quantify protein expression for Kv4.3 and Na+ channel alpha subunits, which were decreased by 72% and 47%, respectively, in P42 dogs (P<0.001 versus control for each), in a manner quantitatively similar to measured changes in mRNA and currents, whereas Na+,Ca2+ exchanger protein concentration was unchanged. We conclude that chronic atrial tachycardia alters atrial ion channel gene expression, thereby altering ionic currents in a fashion that promotes the occurrence of AF. These observations provide a potential molecular basis for the self-perpetuating nature of AF. (+info)
Two regions of sulfonylurea receptor specify the spontaneous bursting and ATP inhibition of KATP channel isoforms.
(30/2415)
KATP channels are heteromultimers of KIR6.2 and a sulfonylurea receptor, SUR, an ATP binding cassette (ABC) protein with several isoforms. KIR6.2 forms a channel pore whose spontaneous activity and ATP sensitivity are modulated by the receptor via an unknown interaction(s). Side by side comparison of single-channel kinetics and steady-state ATP inhibition of human beta-cell, SUR1/KIR6.2, versus cardiac, SUR2A/KIR6.2 channels demonstrate that the latter have a greater mean burst duration and open probability in the absence of nucleotides and approximately 4-fold higher IC50(ATP). We have used matched chimeras of SUR1 and SUR2A to show that the kinetics, which determine the maximal open probability (Pomax), and the ATP sensitivity are functionally separable and to identify the two segments of SUR responsible for these isoform differences. A region within the first five transmembrane domains specifies the interburst kinetics, whereas a C-terminal segment determines the sensitivity to inhibitory ATP. The separable effects of SUR on ATP inhibition and channel kinetics implies that the cytoplasmic C terminus of SUR either directly modulates the affinity of a weak ATP binding site on the inward rectifier or affects linkage between the binding site and the gate. This is the first identification of parts of an ABC protein that interact with an ion channel subunit to modulate the spontaneous activity and ATP sensitivity of the heteromeric channel. (+info)
Distinct molecular bases for pH sensitivity of the guard cell K+ channels KST1 and KAT1.
(31/2415)
Acid-induced potassium uptake through K+ channels is a prerequisite for stomatal opening. Our previous studies identified a pore histidine as a major component of the acid activation mechanism of the potato guard cell K+ channel KST1 (1). Although this histidine is highly conserved among all plant K+ uptake channels cloned so far, the pH-dependent gating of the Arabidopsis thaliana guard cell K+ channel KAT1 was not affected by mutations of this histidine. In both channels, KST1 and KAT1, aspartate mutants in the K+ channel consensus sequence GYGD adjacent to the histidine (KST1-D269N and KAT1-D265N) were inhibited by a rise in the extracellular proton concentration. pH changes affected the half-maximal activation voltage V(1)/(2) of the KST1 mutant, whereas in the mutant channel KAT1-D265N an acid-induced decrease in the maximum conductance gmax indicated the presence of a proton block. In contrast to the wild type KST1, the S4-mutant channel KST1-R181Q exhibited an activation upon alcalization of the extracellular solution. From our electrophysiological studies on channel mutants with respect to the pore histidine as well as the aspartate, we conclude that the common proton-supported shift in the voltage dependence of KST1 and KAT1 is based on distinct molecular elements. (+info)
Suppression of Kir2.3 activity by protein kinase C phosphorylation of the channel protein at threonine 53.
(32/2415)
Kir2.3 plays an important part in the maintenance of membrane potential in neurons and myocardium. Identification of intracellular signaling molecules controlling this channel thus may lead to an understanding of the regulation of membrane excitability. To determine whether Kir2.3 is modulated by direct phosphorylation of its channel protein and identify the phosphorylation site of protein kinase C (PKC), we performed experiments using several recombinant and mutant Kir2.3 channels. Whole-cell Kir2.3 currents were inhibited by phorbol 12-myristate 13-acetate (PMA) in Xenopus oocytes. When the N-terminal region of Kir2.3 was replaced with that of Kir2.1, another member in the Kir2 family that is insensitive to PMA, the chimerical channel lost its PMA sensitivity. However, substitution of the C terminus was ineffective. Four potential PKC phosphorylation sites in the N terminus were studied by comparing mutations of serine or threonine with their counterpart residues in Kir2.1. Whereas substitutions of serine residues at positions 5, 36, and 39 had no effect on the channel sensitivity to PMA, mutation of threonine 53 completely eliminated the channel response to PMA. Interestingly, creation of this threonine residue at the corresponding position (I79T) in Kir2.1 lent the mutant channel a PMA sensitivity almost identical to the wild-type Kir2.3. These results therefore indicate that Kir2.3 is directly modulated by PKC phosphorylation of its channel protein and threonine 53 is the PKC phosphorylation site in Kir2.3. (+info)