Prof Paul Kemp - DPhil (Oxon)
Ion Channels in Respiratory Health and Disease
Our five complementary research interests are in the general areas of molecular physiology, functional genomics and proteomics and are:
a) Regulation and molecular characterization of alveolar ion channels in development, health, and disease (live cell imaging and electrophysiology – Figure 1).
Figure 1. Differential staining of alveolar type I and type II cells within a living lung slice.
A. Confocal image 543nm excitation to visualise Nile Red-positive cells (ATII). 2µM z-scan.
B. Confocal image employing 488nm excitation to visualise mVIIIB2/FITC-positive AT I cells.
C. Digital overlay of the 488nm and 543nm z-scans.
b) Molecular physiology of O 2 and gas transmitter sensing in the human lung and nervous system (K 2P, P2X and BK Ca channels – Figure 2).
Figure 2. Cartoon showing model for hypoxic inhibition of tandem P domain K channels in neuorepithelial body. TASK channel dimer is shown in red. NADPH oxidase is shown in blue.
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c) Transcriptional and translational control of functional expression of human ion channel genes by environmental cues (gene chip arrays and promoter cloning/characterisation – Figure 3).
Figure 3. Ion channel gene chip array showing basal expression levels of ion channels in native untransfected HEK293 cells. An experimental array of 149 DNA sequences (spotted in triplicate), consisting of expressed sequence tags (ESTs) and PCR-amplified, gene-specific sequences, was designed to measure the expression levels of 90 different human cation channel genes (some of which are amplified from more than one EST of the same gene). Total RNA was extracted from HEK293 cells growing under conditions of normoxia and equal amounts of RNA were used to generate Cy3- and Cy5-labelled cDNA. Labelled cDNAs were pooled and hybridized to the array and fluorescence intensities at each spot were measured. Signal intensities were normalized to the b-actin positive control and superimposed using ArrayPro4 software. The results suggest that only a minimal array of cation channels are expressed in native untransfected HEK293 cells.
d) Functional characterisation of human stem cell–derived neural precursors
e) Identification of cellular gas sensors and novel protein partners of gas sensing ion channels (proteomics and loss-of-function approaches - Figure 4).
Figure 4. Proteomics as a method of identifying potassium channel protein partners. The top panels show exemplar 2-D gels of immunoprecipitated proteins as in focused at pH of 3-7 in the horizontal folled by SDS-PAGE in the vertical. The bottom panels show the gels following image analysis. Lower left is the combined matchced set. Of import is the lower right panel which shows the merged image following quantitation and comparison in order to label and distinguish between common (in red) and novel, maxi-K a subunit protein partners (in green).
Major Research Grants & Awards
Awarded & Current
2007-2010 MRC Project Grant
Defining the role of cyclic nucleotide-gated cation channels in lung fluid homeostasis
£630,000 (Principal applicant)
2006-2009 Amgen Project Grant
Role of chronic hypoxia in kidney and lung development and function.
£70, 000. (co-applicant)
2006-2009 BBSRC Project Grant
Role of calcium sensing receptor in lung development
£470,000 (Co-applicant with D Riccardi)
2004-2009 British Heart Foundation Programme Grant
Hypoxic remodelling of ion channels in cardiorespiratory disease: a functional proteomics approach
£567, 587 (principal investigator)