We report the molecular and functional characterization of murine Slc26a6, the putative apical chloride-formate exchanger of the proximal tubule. The Slc26a6 transcript is expressed in several tissues, including kidney. Alternative splicing of the second exon generates two distinct isoforms, denoted Slc26a6a and Slc26a6b, which differ in the inclusion of a 23-residue NH2-terminal extension. Functional comparison with murine Slc26a1, the basolateral oxalate exchanger of the proximal tubule, reveals a number of intriguing differences. Whereas Slc26a6 is capable of Cl−, SO[Formula: see text], formate, and oxalate uptake when expressed in Xenopus laevis oocytes, Slc26a1 transports only SO[Formula: see text] and oxalate. Measurement of intracellular pH during the removal of extracellular Cl− in the presence and absence of HCO[Formula: see text] indicates that Slc26a6 functions as both a Cl−/HCO[Formula: see text] and a Cl−/OH− exchanger; simultaneous membrane hyperpolarization during these experimental maneuvers reveals that HCO[Formula: see text] and OH− transport mediated by Slc26a6 is electrogenic. Cis-inhibition and efflux experiments indicate that Slc26a6 can mediate the exchange of both Cl− and SO[Formula: see text]with a number of substrates, including formate and oxalate. In contrast, SO[Formula: see text] and oxalate transport by Slc26a1 are mutually cis-inhibited but activated significantly by extracellular halides, lactate, and formate. The data indicate that Slc26a6 encodes an apical Cl−/formate/oxalate and Cl−/base exchanger and reveal significant mechanistic differences between apical and basolateral oxalate exchangers of the proximal tubule.
Peripheral neuropathy associated with agenesis of the corpus callosum (ACCPN) is a severe sensorimotor neuropathy associated with mental retardation, dysmorphic features and complete or partial agenesis of the corpus callosum. ACCPN is transmitted in an autosomal recessive fashion and is found at a high frequency in the province of Quebec, Canada. ACCPN has been previously mapped to chromosome 15q. The gene SLC12A6 (solute carrier family 12, member 6), which encodes the K+-Cl- transporter KCC3 and maps within the ACCPN candidate region, was screened for mutations in individuals with ACCPN. Four distinct protein-truncating mutations were found: two in the French Canadian population and two in non-French Canadian families. The functional consequence of the predominant French Canadian mutation (2436delG, Thr813fsX813) was examined by heterologous expression of wildtype and mutant KCC3 in Xenopus laevis oocytes; the truncated mutant is appropriately glycosylated and expressed at the cellular membrane, where it is non-functional. Mice generated with a targeted deletion of Slc12a6 have a locomotor deficit, peripheral neuropathy and a sensorimotor gating deficit, similar to the human disease. Our findings identify mutations in SLC12A6 as the genetic lesion underlying ACCPN and suggest a critical role for SLC12A6 in the development and maintenance of the nervous system.
Evaluation of candidate loci culminated in the identification of a heterozygous missense mutation (R67W) in KCNJ2, the gene encoding the inward-rectifying potassium current, Kir2.1, in 41 members of a kindred in which ventricular arrhythmias (13 of 16 female members [81%]) and periodic paralysis (10 of 25 male members [40%]) segregated as autosomal dominant traits with sex-specific variable expressivity. Some mutation carriers exhibited dysmorphic features, including hypertelorism, small mandible, syndactyly, clinodactyly, cleft palate, and scoliosis, which, together with cardiodysrhythmic periodic paralysis, have been termed "Andersen syndrome." However, no individual exhibited all manifestations of Andersen syndrome, and this diagnosis was not considered in the proband until other family members were examined. Other features seen in this kindred included unilateral dysplastic kidney and cardiovascular malformation (i.e., bicuspid aortic valve, bicuspid aortic valve with coarctation of the aorta, or valvular pulmonary stenosis), which have not been previously associated. Nonspecific electrocardiographic abnormalities were identified in some individuals, but none had a prolonged QT interval. Biophysical characterization of R67W demonstrated loss of function and a dominant-negative effect on Kir2.1 current. These findings support the suggestion that, in addition to its recognized role in function of cardiac and skeletal muscle, KCNJ2 plays an important role in developmental signaling.
KCNE1, also known as minK, is a member of the KCNE family of membrane proteins that modulate the function of KCNQ1 and certain other voltage-gated potassium channels (K V ). Mutations in human KCNE1 cause congenital deafness and congenital long QT syndrome, an inherited predisposition to potentially life-threatening cardiac arrhythmias. Although its modulation of KCNQ1 function has been extensively characterized, many questions remain regarding KCNE1's structure and location within the channel complex. In this study KCNE1 was overexpressed in E. coli and purified. Micellar solutions of the protein were then microinjected into Xenopus oocytes expressing KCNQ1 channels, followed by electrophysiological recordings to test whether recombinant KCNE1 can co-assemble with the channel. Native-like modulation of channel properties was observed following injection of KCNE1 in lysomyristoylphosphatidylglycerol (LMPG) micelles, indicating that KCNE1 is not irreversibly misfolded and that LMPG is able to act as a vehicle for delivering membrane proteins into the membranes of viable cells. 1 H, 15 N-TROSY NMR experiments indicated that LMPG micelles are well-suited for structural studies of KCNE1, leading to assignment of its backbone resonances and to relaxation studies. The chemical shift data confirmed that KCNE1's secondary structure includes several α-helices and demonstrated that its distal Cterminus is disordered. Surprisingly, for KCNE1 in LMPG micelles there appears to be a break in α-helicity at sites 59−61, near the middle of the transmembrane segment, a feature that is accompanied by increased local backbone mobility. Given that this segment overlaps with sites 57 −59, which are known to play a critical role in modulating KCNQ1 channel activation kinetics, this unusual structural feature is likely of considerable functional relevance.Voltage-gated potassium channels (K V ) play a variety of important roles in human health and disease. For example, human KCNQ1 is essential to the cardiac action potential that mediates heartbeat and is also critical for potassium ion homeostasis in the inner ear(1;2). The function of several K V channels is modulated by accessory proteins including K V channel β subunits (Kvβ)(3-6), potassium channel interacting proteins (KCHiP)(7;8), and the KCNE family of † This study was supported by NIH grant R01 DC007416 to CRS, NIH grant HL077188 to ALG, a Vanderbilt School of Medicine Discovery Grant to CGV, and by a postdoctoral fellowship from the American Heart Association to CT (0625586B). Some data were collected in the SEC NMR facility at the University of Georgia, which is supported by US NIH grant P41 GM066340. The NMR assignments for KCNE1 that are reported in this paper have been deposited in the BMRB with access number 15102. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript single transmembrane proteins including KCNE1 and minK-related peptides (MiRPs) (9)(10)(11)(12)(13)(14). KCNE1, also known as minK, co-assembles with KCNQ1 in heart muscle cells...
KCNE1 (minK), found in the human heart and, cochlea is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of POPC and POPG was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (KV7.1) voltage-gated potassium channel led to native-like modulation of the channel. CD spectroscopy demonstrated that the %-helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles relative to the previously described structure in 1.0% LMPG micelles. SDSL EPR spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu 59, and Ser64 KCNE1 in both POPC/POPG vesicles and in LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu 59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water-inaccessible in vesicles, but found to be water-accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers, but also demonstrates the need to refine this structure using data derived from bilayer-reconstituted protein in order to more accurately define its native structure. This work establishes the basis for such future studies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.