Cell swelling activates ATP-dependent voltage-gated chloride channels in M-1 mouse cortical collecting duct cells.
In the present study we used whole-cell patch clamp recordings to investigate swelling-activated Cl-currents (ICl-swell) in M-1 mouse cortical collecting duct (CCD) cells. Hypotonic cell swelling reversibly increased the whole-cell Cl- conductance by about 30-fold. The I-V relationship was outwardly-rectifying and ICl-swell displayed a characteristic voltage-dependence with relatively fast inactivation upon large depolarizing and slow activation upon hyperpolarizing voltage steps. Reversal potential measurements revealed a selectivity sequence SCN- > I- > Br- > Cl- > > gluconate. ICl-swell was inhibited by tamoxifen, NPPB (5-nitro-2(3-phenylpropylamino)-benzoate), DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid), flufenamic acid, niflumic acid, and glibenclamide, in descending order of potency. Extracellular cAMP had no significant effect. ICl-swell was Ca2+ independent, but current activation depended on the presence of a high-energy gamma-phosphate group from intracellular ATP or ATP gamma S. Moreover, it depended on the presence of intracellular Mg2+ and was inhibited by staurosporine, which indicates that a phosphorylation step is involved in channel activation. Increasing the cytosolic Ca2+ concentration by using ionomycin stimulated Cl- currents with a voltage dependence different from that of ICl-swell. Analysis of whole-cell current records during early onset of ICl-swell and during final recovery revealed discontinuous step-like changes of the whole-cell current level which were not observed under nonswelling conditions. A single-channel I-V curve constructed using the smallest resolvable current transitions detected at various holding potentials and revealed a slope conductance of 55, 15, and 8 pS at +120, 0, and -120 mV, respectively. The larger current steps observed in these recordings had about 2, 3, or 4 times the size of the putative single-channel current amplitude, suggesting a coordinated gating of several individual channels or channel subunits. In conclusion we have functionally characterized ICl-swell in M-1 CCD cells and have identified the underlying single channels in whole-cell current recordings.
Meyer
K
K
Zentrum der Physiologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Korbmacher
C
C
D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D048429:Cell Size; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D004594:Electrophysiology; D005110:Extracellular Space / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007038:Hypotonic Solutions; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007672:Kidney Cortex / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D018408:Patch-Clamp Techniques
D018118:Chloride Channels; D007038:Hypotonic Solutions; D000255:Adenosine Triphosphate; D002118:Calcium