Elektrophysiologie / Electrophysiology (Leitung: Prof. Dr.
Wolf-Michael Weber)
Specific inhibition of ENaC expressed in Xenopus laevis oocytes by antisense oligonuleotides (Project 5)
We expressed the three subunits of the epithelial Na+ channel (ENaC) from rat colon in oocytes of the South African clawed toad Xenopus laevis. The
objective of this investigation was to find an effective long lasting block of ENaC with respect to improve the present amiloride therapy for cystic fibrosis patients. We
searched for specific regions on the ENaC sequence that are required for functional expression of the channel. These regions were used to construct antisense
oligonucleotides (AONs) that prevent ENaC expression by specific binding to the ENaC mRNA, thereby stopping complete translation. AONs are short sequences of DNA
designed to inhibit the expression of a protein by specifically binding to its mRNA. We designed two such AONs with sequences complementary to the mRNA of both rat
and human αENaC subunit. Special software was used to compute predicted secondary structures of rat (r) and human (h) αENaC mRNA at different
temperatures ranging from 12 to 36°C. We then searched for sequences of 16 bases that are common to the two species and are also mostly single-stranded. The two
AONs we termed green AON (gAON) and black AON (bAON), correspond to hENaC at positions 290 and 1670, respectively, and to αrENaC mRNA 347 and
1733. The actual molecules were synthesized as 2OMeRNA phosphorothioates. Co-injection of either AON with αβγrENaC in oocytes successfully
inhibits channel expression, as demonstrated by the negative membrane potential (Vm) and the lack of amiloride sensitivity of the clamp current
(Im) and conductance (Gm). Sequential microinjection of ENaC mRNA followed by either AON led to an exponential-like decrease in
amiloride-sensitive current (INa) and conductance (GNa), with highly variable time constants ranging from 2 hours to 2 days, accompanied by a
gradual shift of the membrane potential from positive to negative values. Pooled data from 10 oocytes shows for gAON a decrease in INa from 955 nA before
AON injection to 814 nA on day 1 and 352 nA on day 2 after AON injection, while GNa decreases from 13.9 microS (day 0) to 11.5 microS (day 1) and 5.5 microS (day 2). For bAON the data (16 oocytes) are: INa from 4393 nA (day 0) to 421 nA (day 1); GNa from 69.3 microS to 6.9 microS;
Vm from 10.4 mV to 11.3 mV. Our data are a first step in direction of a clinical test with AON-containing aerosol to reduce Na+
hyperabsorption in CF epithelia.
More information: http://www.uni-muenster.de/Biologie.Zoophysiologie/electrophys/ElectroHome.htm
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