Abstract
Mutations in either KCNQ2 or KCNQ3 cause benign familial neonatal convulsions (BFNC), an inherited epilepsy of newborns. Both proteins are
co-expressed in the brain and associate to form heteromeric potassium channels. They are thought to give rise to the so called M-currents, which are important
regulators of neuronal excitability.
In this work the basis for the current increase seen upon co-expression of both KCNQ subunits in Xenopus oocytes was investigated. Therefore the
single-channel conductances and open-probabilities of homo- and heteromeric KCNQ2 and KCNQ3 channels were determined ((KCNQ2: i» 18pS, popen»
0,61) (KCNQ2/KCNQ3: i» 16pS, popen» 0,72) (KCNQ3: i » 7,3, popen» 0,42)). The mean single channel conductance and popen of heteromeric channels
were not significantly different from those of homomeric KCNQ2 channels, whereas the conductance of KCNQ3 was about half that value. This indicates that
the increase of currents observed upon co-expressing both subunits in Xenopus oocytes cannot be explained by an increase in these parameters.
Co-expression with KCNQ3 increased the surface expression of KCNQ2 by a factor of 5, and co-expression of KCNQ3 with KCNQ2 led to a >10fold
increase of KCNQ3 surface expression. A KCNQ2 mutant associated with BFNC that truncates the cytoplasmic carboxyterminus neither reached the surface
nor stimulated KCNQ3 surface expression. By contrast, BFNC missense mutations in the transmembrane regions of KCNQ2 or KCNQ3 left surface
expression unchanged. Thus, the increase in currents seen upon co-expressing KCNQ2 and KCNQ3 is predominantly due to an increase in surface
expression, which is dependent on an intact carboxyterminus.
In the kidney ClC-5 chloride channel resides mainly in vesicles of the endocytotic pathway and contributes to their acidification. Its disruption in mice entails a
broad defect in renal endocytosis and causes secondary changes in calciotropic hormone levels. Inactivating mutations in Dent?s disease lead to proteinuria and
kidney stones. Possibly by recycling, a small fraction of ClC-5 also reaches the plasma membrane. We identified a carboxy-terminal internalization motif in
ClC-5. It resembles the so called PY motif which is crucial for the endocytosis and degradation of epithelial Na+-channels (ENaC). Disrupting this motif
increases surface expression and currents about 2-fold. This is likely due to interactions with WW-domains as dominant negative mutants of the
ubiquitin-protein ligase WWP2 increased surface expression and currents of ClC-5 only when its PY motif was intact. Stimulating endocytosis by expressing
rab5 or its GTPase-deficient Q79L mutant decreased WT ClC-5 currents, but did not affect channels with mutated motifs. Similarly, decreasing endocytosis
by expressing the inactive S34N mutant of rab5 increased ClC-5 currents only if its PY motif was intact. Thus, the endocytosis of ClC-5, which itself is crucial
for the endocytosis of other proteins, depends on the interaction of a carboxy-terminal internalization signal with ubiquitin-protein ligases containing
WW-domains. |
