Biomedical subjects

Martina Knirsch

Publications and source records attributed to Martina Knirsch.

6 recordsLinked to original sources

PubMed2007

Large-scale mapping of mutations affecting zebrafish development.

BACKGROUND: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. RESULTS: We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. CONCLUSION: By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.

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PubMed2006

Functional coassembly of KCNQ4 with KCNE-beta- subunits in Xenopus oocytes.

The KCNQ gene family comprises voltage-gated potassium channels expressed in epithelial tissues (KCNQ1, KCNQ5), inner ear structures (KCNQ1, KCNQ4) and the brain (KCNQ2-5). KCNQ4 is expressed in inner and outer hair cells of the inner ear where it influences electrical excitability and cell survival. Accordingly, loss of function mutations of the KCNQ4 gene cause hearing loss in humans and functional k.o.-mice show progressive degeneration of outer hair cells (OHCs). However, characteristic electrophysiological features of the native KCNQ4- carried current I(K,n) in OHCs are not recapitulated by expression of KCNQ4 channels in heterologous expression systems. This might suggest modulation of KCNQ4 by interacting KCNE Beta-subunits, which are known to modify the properties of the closely related KCNQ1. The present study explored whether transcripts of the KCNE isoforms could be identified in OHC mRNA and whether the subunits modulate KCNQ4 function. RT-PCR indeed yielded transcripts of all five KCNEs in OHCs. Coexpression of the KCNE- Beta-subunits with human KCNQ4 in the Xenopus laevis oocyte expression system revealed that all KCNEs modulate KCNQ4 voltage dependence, protein stability and ion selectivity of hKCNQ4 in Xenopus oocytes. The deafness-associated Jervell and Lange- Nielsen syndrome (JLNS) mutation KCNE1(D76N) impairs KCNQ4-function whereas the Romano-Ward syndrome (RWS) mutant KCNE1(S74L), which shows normal hearing in patients, does not impair KCNQ4 channel function. In conclusion, KCNEs are presumably coexpressed with KCNQ4 in hair cells from the organ of Corti and might regulate KCNQ4 functional properties, effects that could be important under physiological and pathophysiological conditions.

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PubMed2006

Thyroid hormone receptors TRalpha1 and TRbeta differentially regulate gene expression of Kcnq4 and prestin during final differentiation of outer hair cells.

Thyroid hormone (TH or T3) and TH-receptor beta (TRbeta) have been reported to be relevant for cochlear development and hearing function. Mutations in the TRbeta gene result in deafness associated with resistance to TH syndrome. The effect of TRalpha1 on neither hearing function nor cochlear T3 target genes has been described to date. It is also uncertain whether TRalpha1 and TRbeta can act simultaneously on different target genes within a single cell. We focused on two concomitantly expressed outer hair cell genes, the potassium channel Kcnq4 and the motor protein prestin Slc26a5. In outer hair cells, TH enhanced the expression of the prestin gene through TRbeta. Simultaneously Kcnq4 expression was activated in the same cells by derepression of TRalpha1 aporeceptors mediated by an identified THresponse element, which modulates KCNQ4 promoter activity. We show that T3 target genes can differ in their sensitivity to TH receptors having the ligand either bound (holoreceptors) or not bound (aporeceptors) within single cells, and suggest a role for TRalpha1 in final cell differentiation.

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PubMed2005

Regulation of KCNQ4 potassium channel prepulse dependence and current amplitude by SGK1 in Xenopus oocytes.

The KCNQ gene family comprises voltage-gated potassium channels expressed in epithelial tissues (KCNQ1, KCNQ5), inner ear structures (KCNQ1, KCNQ4) and the brain (KCNQ2-5). KCNQ4 is expressed in inner and outer hair cells of the inner ear where it determines electrical excitability. Accordingly, loss of function mutations of the KCNQ4 gene cause hearing loss. Several K+ channels including the closely related KCNQ1/KCNE1 channel are regulated by the serum- and glucocorticoid-inducible kinase (SGK) family. The present study utilized the Xenopus oocyte system to explore effects of SGK isoforms on KCNQ4 mediated K(+)-currents: KCNQ4 channels activated in a voltage dependent manner with half maximal activation at -10 mV. The peak channel activity was significantly increased by prepulsing. Coexpression of wild type SGK1 but not coexpression of the inactive mutant (K127N)SGK1 significantly increased current amplitudes (by 67 %) and significantly increased the resting potential of KCNQ4 expressing oocytes. Here we describe for the first time a prepulse dependence of KCNQ4 channels with increased currents after hyperpolarizing prepulses. Coexpression of SGK1 significantly attenuated the effect of prepulsing on peak currents. Mutation of Ser to Asp or Ala in the putative phosphorylation consensus sequence in KCNQ4 significantly decreased the sensitivity to SGK1-coexpression. In conclusion, SGK1 regulates current amplitudes and kinetic properties of KCNQ4 channel activity, an effect sensitive to mutations in the SGK1 consensus sequence of the channel.

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PubMed2004

Deletion of the Ca2+-activated potassium (BK) alpha-subunit but not the BKbeta1-subunit leads to progressive hearing loss.

The large conductance voltage- and Ca2+-activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming alpha-subunit coexpressed with the auxiliary beta1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel alpha-(BKalpha-/-) and beta1-subunit (BKbeta1-/-) knockout mice, we demonstrate normal hearing function and cochlear structure of BKbeta1-/- mice. During the first 4 postnatal weeks also, BKalpha-/- mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKalpha-/- mice only from approximately 8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations.

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PubMed2003

Cav1.3 (alpha1D) Ca2+ currents in neonatal outer hair cells of mice.

Outer hair cells (OHC) serve as electromechanical amplifiers that guarantee the unique sensitivity and frequency selectivity of the mammalian cochlea. It is unknown whether the afferent fibres connected to adult OHCs are functional. If so, voltage-activated Ca2+ channels would be required for afferent synaptic transmission. In neonatal OHCs, Ca2+ channels seem to play a role in maturation since OHCs from Cav1.3-deficient (Cav1.3-/-) mice degenerate shortly after the onset of hearing. We therefore studied whole-cell Ca2+ currents in outer hair cells aged between postnatal day 1 (P1) and P8. OHCs showed a rapidly activating inward current that was 1.8 times larger with 10 mM Ba2+ as charge carrier (IBa) than with equimolar Ca2+ (ICa). IBa started activating at -50 mV with Vmax = -1.9 +/- 6.9 mV, V0.5 = -15.0 +/- 7.1 mV and k = 8.2 +/- 1.1 mV (n = 34). The peak IBa showed negligible inactivation (3.6 % after 300 ms) whereas the ICa (10 mM Ca2+) was inactivated by 50.7 %. OHC IBa was reduced by 33.5 +/- 10.3 % (n = 14) with 10 microM nifedipine and increased to 178.5 +/- 57.8 % (n = 14) by 5 microM Bay K 8644. A dose-response curve for nifedipine revealed an IC50 of 2.3 microM, a Hill coefficient of 2.7 and a maximum block of 36 %. Average IBa density in OHCs was 24.4 +/- 10.8 pA pF-1 (n = 105) which is only 38 % of the value in inner hair cells. Single cell RT-PCR revealed expression of Cav1.3 in OHCs. In OHCs from Cav1.3-/- mice, Ba2+ current density was reduced to 0.6 +/- 0.5 pA pF-1 (n = 9) indicating that > 97 % of the Ca2+ channel current in OHCs flows through Cav1.3.

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