Cl- channels in basolateral TAL membranes XV. Molecular heterogeneity between cortical and medullary channels

C. J. Winters, L. Zimniak, M. V. Mikhailova, W. B. Reeves, T. E. Andreoli

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

We have isolated two new and highly homologous cDNAs, mmClC-Ka from mouse outer medulla and mcClC-Ka from mouse cortex. In both cases, mRNA was obtained from the indicated region and subjected to RT-PCR using primers from the nucleotide sequence of rbClC-Ka, which encodes basolateral Cl- channels (termed rbClC-Ka) in rabbit MTAL. The predicted protein products of mmClC-Ka and mcClC-Ka, mmClC-Ka and mcClC-Ka, respectively, were 85% homologous and had predicted molecular weights of 75 kDa. The predicted protein sequences for mmClC-Ka and rbClC-Ka had three cytosolic sites - threonine 185, threonine 187 and serine 270 - which were absent in mcClC-Ka. These three moieties represent potential sites for phosphorylation of mmClC-Ka and rbClC-Ka, but not of mc-ClC-Ka, and may account for the failure of (ATP + PKA) to increase the open time probability Po in basolateral CTAL Cl- channels. We prepared antisense oligonucleotides specific for nonhomologous regions of these two cDNAs, mmAnti-sense for mmClC-Ka and mcAntisense for mcClC-Ka. Using anti-rbClC-Ka, a polyclonal antibody to rbClC-Ka, we found that, when transfected into cultured mouse MTAL and CTAL cells, mmAntisense suppressed the appearance of the 75 kDa band by 50% in vesicles from MTAL but not CTAL cells, while transfection of MTAL and CTAL cells with mcAntisense suppressed appearance of the 75 kDa band in vesicles from CTAL but not MTAL cells, mmAntisense transfection also prolonged the half-time (T1/2, sec) for 36Cl- efflux in cultured MTAL cells from 82.4 ± 6.8 sec (SEM) to 187.8 ± 9.5 sec (n = 5; P = 0.0001) while mcAntisense transfection had no such effect. Conversely, in cultured CTAL cells, mcAntisense transfection prolonged the T1/2 for 36Cl- efflux from 80.9 ± 6.3 sec to 191.8 ± 6.5 sec (n = 5; P = 0.00005), while mmAntisense had no such effect. We conclude that mmClC-Ka and mcClC-Ka may encode the basolateral Cl- channels mediating net Cl- absorption in mouse MTAL and CTAL, respectively.

Original languageEnglish (US)
Pages (from-to)221-230
Number of pages10
JournalJournal of Membrane Biology
Volume177
Issue number3
DOIs
StatePublished - Oct 1 2000
Externally publishedYes

Keywords

  • Basolateral membranes
  • Cl channels
  • Molecular heterogeneity
  • Thick limbs

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

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