HYPERTONIA ÉS NEPHROLOGIA - eLitMed.hu

2002; 6 (1):3–15. ONTOGENY OF POTASSIUM TRANSPORT IN THE DISTAL NEPHRON 9
and density of conducting apical K + channels in principal cells
(19).
The predominant K + permeable channel in cell-attached
patches of the apical membrane of the principal cell is a
low-conductance (30-40 pS) inwardly-rectifying ATP-sensitive
secretory K + (SK) channel (1; 18; 44; 45). The prevalence
of this channel and its high Po at the resting membrane
potential suggests that this channel most likely mediates K +
secretion under baseline conditions. Patch clamp analysis of
maturing rabbit principal cells identified an increase in the
mean number of conducting stock channels per patch after the
second week of life (Fig. 5) (19), approximately one week
after an increase in activity of the amiloride-sensitive Na +
channel (ENaC) was identified (Fig. 5) (26). Conducting
apical stock channels were not detected in CCDs isolated from
animals in the first week of postnatal life. The increase in
number of conducting channels appeared to be due primarily
to a developmental increase in number of channels in each
cell-attached patch (19). Although the possibility of an early
increase in Po of existing channels could not be excluded, the
Po of the SK channel did not change between 3 and 5 weeks of
age, a time interval during which the number of conducting
channels increased almost 3-fold (Fig. 5) (19). As the SK
channel constitutes the rate-limiting step for K + secretion in
fully differentiated CCD, we speculate that the limited
capacity of the immature CCD for K + secretion is due, at least
partially, to a scarcity of conducting SK channels in the
connecting and collecting ducts.
% of adult (6 wk)
ROMK mRNA
abundance
120
100
80
60
40
20
0
0 1 2 3 4 5 6 7
age (wks)
ROMK was originally cloned from rat outer medulla where
an apical low-conductance KATP channel in the thick
ascending limb of the loop of Henle (TALH) recycles K +
across the apical membrane to ensure an abundant supply of
substrate for the bumetanide-sensitive Na-K-2Cl cotransporter
(46). The observation that ROMK encodes a family of
ATP-sensitive K + channels with biophysical and regulatory
properties similar to those of the SK channel (47-49) provides
compelling evidence that ROMK represents the major
functional subunit of the SK channel. Multiple ROMK
isoforms have been identified in rat and human kidney
(47-51). All isoforms, derived from alternative splicing of the
5’ end of the gene, share a common core exon that encodes
more than 95% of the channel protein sequence; they differ
only in the sequence and length of their amino termini. When
expressed in Xenopus laevis oocytes, ROMK1, 2 and 3 lead to
expression of similar K + -selective, barium-sensitive inwardly
rectifying channels (48; 49; 51).
To test whether the postnatal increase in number of
conducting SK channels in the CCD is mediated by a
developmental increase in transcription ROMK, we used
gene-specific molecular probes to examine the steady state
expression of ROMK mRNA in maturing rabbit kidneys and,
more specifically, the developmental appearance of ROMK
message in single CCDs. Northern analysis of mRNA isolated
from rat and rabbit kidney revealed a single band of expected
size for ROMK (~3.1 and 2.9 kb, respectively) at all ages (32;
52). Densitometric analysis revealed a developmental
Figure 7. Maturational increase in ROMK mRNA expression in rat and rabbit. Thirty µg of total RNA from kidneys of
1- to 6-wk-old animals rats were fractionated on a denaturing formaldehyde gel and transferred to a nylon filter.
Hybridization with the 32 P-labeled ROMK probe revealed a single transcript at ~3 kb. The Northern blot was then
rehybridized to a -actin probe as a loading and transfer control. Laser densitometry was used to obtain ROMK/
-actin mRNA ratios in 4 blots prepared from each species. For each blot, the ratios were expressed as a percentage of
the value obtained at 6 weeks, the latter set to equal 100%. ROMK mRNA abundance increased significantly during
postnatal life in both rat and rabbit (p