Jennifer J. Paynter

Bio: Jennifer J. Paynter is an academic researcher from University of Oxford. The author has contributed to research in topics: Gating & Complementation. The author has an hindex of 5, co-authored 5 publications receiving 109 citations.

Control of pH and PIP2 Gating in Heteromeric Kir4.1/Kir5.1 Channels by H-Bonding at the Helix-Bundle Crossing

Abstract: Inhibition by intracellular H(+) (pH gating) and activation by phosphoinositides such as PIP(2) (PIP(2)-gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP(2) gating and pH gating are controlled by an intra-subunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP(2) and pH gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP2 gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP(2) affinities. This indicates that Kir channel PIP(2) affinity has little impact on either the PIP(2) or pH gating kinetics.

Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels.

Abstract: Our understanding of the mammalian inwardly rectifying family of K+ channels (Kir family) has recently been advanced by X-ray crystal structures of two homologous prokaryotic orthologs (KirBac1.1 a...

Random mutagenesis screening indicates the absence of a separate H(+)-sensor in the pH-sensitive Kir channels.

Abstract: Several inwardly-rectifying (Kir) potassium channels (Kir1.1, Kir4.1 and Kir4.2) are characterised by their sensitivity to inhibition by intracellular H(+) within the physiological range. The mechanism by which these channels are regulated by intracellular pH has been the subject of intense scrutiny for over a decade, yet the molecular identity of the titratable pH-sensor remains elusive. In this study we have taken advantage of the acidic intracellular environment of S. cerevisiae and used a K(+) -auxotrophic strain to screen for mutants of Kir1.1 with impaired pH-sensitivity. In addition to the previously identified K80M mutation, this unbiased screening approach identified a novel mutation (S172T) in the second transmembrane domain (TM2) that also produces a marked reduction in pH-sensitivity through destabilization of the closed-state. However, despite this extensive mutagenic approach, no mutations could be identified which removed channel pH-sensitivity or which were likely to act as a separate H(+) -sensor unique to the pH-sensitive Kir channels. In order to explain these results we propose a model in which the pH-sensing mechanism is part of an intrinsic gating mechanism common to all Kir channels, not just the pH-sensitive Kir channels. In this model, mutations which disrupt this pH-sensor would result in an increase, not reduction, in pH-sensitivity. This has major implications for any future studies of Kir channel pH-sensitivity and explains why formal identification of these pH-sensing residues still represents a major challenge.

Genetic selection of activatory mutations in KcsA.

Abstract: The KcsA potassium channel from Streptomyces lividans is one of the most actively studied ion channels. However, there are still unresolved issues about its gating mechanism in vivo because the channel is only activated by highly acidic intracellular pH, meaning that it will be mostly inactive in its host environment. In this study we have used a genetic complementation assay of K+-auxotrophic E. coli (TK2420) and S. cerevisiae (SGY1528) to identify activatory or 'gain-of-function' mutations which allow functional activity of KcsA in the physiological environment of two markedly different expression systems. These mutations clustered at the helix-bundle-crossing in both TM1 and TM2 (residues H25, L105, A108, T112, W113, F114, E118 and Q119), and include residues previously implicated in the pH-gating mechanism. We discuss how these gain-of-function mutations may result in their activatory phenotype, the relative merits of the E. coli and S. cerevisiae genetic complementation approaches for the identification of gating mutations in prokaryotic K+ channels, and ways in which this assay may be improved for future use in screening protocols.

Seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME syndrome) caused by mutations in KCNJ10

Abstract: We describe members of 4 kindreds with a previously unrecognized syndrome characterized by seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (hypokalemia, metabolic alkalosis, and hypomagnesemia). By analysis of linkage we localize the putative causative gene to a 2.5-Mb segment of chromosome 1q23.2-23.3. Direct DNA sequencing of KCNJ10, which encodes an inwardly rectifying K(+) channel, identifies previously unidentified missense or nonsense mutations on both alleles in all affected subjects. These mutations alter highly conserved amino acids and are absent among control chromosomes. Many of these mutations have been shown to cause loss of function in related K(+) channels. These findings demonstrate that loss-of-function mutations in KCNJ10 cause this syndrome, which we name SeSAME. KCNJ10 is expressed in glia in the brain and spinal cord, where it is believed to take up K(+) released by neuronal repolarization, in cochlea, where it is involved in the generation of endolymph, and on the basolateral membrane in the distal nephron. We propose that KCNJ10 is required in the kidney for normal salt reabsorption in the distal convoluted tubule because of the need for K(+) recycling across the basolateral membrane to enable normal activity of the Na(+)-K(+)-ATPase; loss of this function accounts for the observed electrolyte defects. Mice deficient for KCNJ10 show a related phenotype with seizures, ataxia, and hearing loss, further supporting KCNJ10's role in this syndrome. These findings define a unique human syndrome, and establish the essential role of basolateral K(+) channels in renal electrolyte homeostasis.

KCNJ10 gene mutations causing EAST syndrome (epilepsy, ataxia, sensorineural deafness, and tubulopathy) disrupt channel function.

Abstract: Mutations of the KCNJ10 (Kir4.1) K+ channel underlie autosomal recessive epilepsy, ataxia, sensorineural deafness, and (a salt-wasting) renal tubulopathy (EAST) syndrome. We investigated the localization of KCNJ10 and the homologous KCNJ16 in kidney and the functional consequences of KCNJ10 mutations found in our patients with EAST syndrome. Kcnj10 and Kcnj16 were found in the basolateral membrane of mouse distal convoluted tubules, connecting tubules, and cortical collecting ducts. In the human kidney, KCNJ10 staining was additionally observed in the basolateral membrane of the cortical thick ascending limb of Henle's loop. EM of distal tubular cells of a patient with EAST syndrome showed reduced basal infoldings in this nephron segment, which likely reflects the morphological consequences of the impaired salt reabsorption capacity. When expressed in CHO and HEK293 cells, the KCNJ10 mutations R65P, G77R, and R175Q caused a marked impairment of channel function. R199X showed complete loss of function. Single-channel analysis revealed a strongly reduced mean open time. Qualitatively similar results were obtained with coexpression of KCNJ10/KCNJ16, suggesting a dominance of KCNJ10 function in native renal KCNJ10/KCNJ16 heteromers. The decrease in the current of R65P and R175Q was mainly caused by a remarkable shift of pH sensitivity to the alkaline range. In summary, EAST mutations of KCNJ10 lead to impaired channel function and structural changes in distal convoluted tubules. Intriguingly, the metabolic alkalosis present in patients carrying the R65P mutation possibly improves residual function of KCNJ10, which shows higher activity at alkaline pH.

Ion Channels in Microbes

Abstract: Studies of ion channels have for long been dominated by the animalcentric, if not anthropocentric, view of physiology. The structures and activities of ion channels had, however, evolved long before the appearance of complex multicellular organisms on earth. The diversity of ion channels existing in cellular membranes of prokaryotes is a good example. Although at first it may appear as a paradox that most of what we know about the structure of eukaryotic ion channels is based on the structure of bacterial channels, this should not be surprising given the evolutionary relatedness of all living organisms and suitability of microbial cells for structural studies of biological macromolecules in a laboratory environment. Genome sequences of the human as well as various microbial, plant, and animal organisms unambiguously established the evolutionary links, whereas crystallographic studies of the structures of major types of ion channels published over the last decade clearly demonstrated the advantage of using microbes as experimental organisms. The purpose of this review is not only to provide an account of acquired knowledge on microbial ion channels but also to show that the study of microbes and their ion channels may also hold a key to solving unresolved molecular mysteries in the future.

Structure of a KirBac potassium channel with an open bundle crossing indicates a mechanism of channel gating

Abstract: KirBac channels are the bacterial homologs of mammalian inwardly rectifying potassium (Kir) channels. All available structures are closed at the helix bundle crossing, but the crystal structure of an open-state KirBac channel now indicates how opening of the primary activation gate may be physically coupled to a rotational twist in the cytoplasmic domain.