Deranged Kv channel regulation in fibroblasts from mice lacking the serum and glucocorticoid inducible kinase SGK1.
Coexpression of the serum and glucocorticoid inducible kinase 1 (SGK1) up-regulates Kv channel activity in Xenopus oocytes and human embryonic kidney cells. To investigate the physiological impact of SGK1 dependent Kv channel regulation, we recorded whole-cell currents in lung fibroblasts from SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+). Serum-grown mouse lung fibroblasts (MLF) from both genotypes exhibited voltage-gated outwardly rectifying K(+)-currents with time-dependent activation (tau(act) approximately 3 msec), slow inactivation (tau(inact) approximately 700 msec), use-dependent inactivation, and (partial) inhibition by K(+) channel blockers TEA, 4-AP, and margatoxin. In serum grown MLF peak Kv current density at +100 mV was significantly lower in sgk1-/- (14 +/- 2 pA/pF, n = 13) than in sgk1+/+ (31 +/- 4 pA/pF, n = 16). PCR amplification of different Kv1 and Kv3 subunits from mouse fibroblasts demonstrated the expression of Kv1.1-1.7, Kv3.1, and Kv3.3 mRNA in both sgk1+/+ and sgk1-/- cells. Upon serum deprivation Kv currents almost disappeared in sgk1+/+ (4 +/- 1 pA/pF, n = 11) but not in sgk1-/- (10 +/- 1 pA/pF, n = 6) MLF. Accordingly, following serum deprivation Kv current density was significantly lower in sgk1+/+ than in sgk1-/-. Stimulation of serum-depleted cells with dexamethasone (dex) (1 microM, 1 day), IGF-1 (6.7 microM, 4-6 h) or both, significantly activated Kv currents in sgk1+/+ but not in sgk1-/- MLF. In the presence of both, dex and IGF-1, the Kv current density was significantly larger in sgk1+/+ (27 +/- 3 pA/pF, n = 12) than in sgk1-/- (13 +/- 3 pA/pF, n = 10) cells. Similar to MLF, Kv currents were significantly higher in sgk1+/+ mouse tail fibroblasts (MTF). In sgk1+/+ but not sgk1-/- MTF the Kv currents were inhibited upon serum deprivation and reincreased after stimulation of serum deprived MTF with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h). According to Fura-2-fluorescence capacitative Ca(2+) entry was lower in sgk1-/- MTF compared to sgk1+/+ MTF. Upon serum deprivation capacitative Ca(2+) entry decreased significantly in sgk1+/+ but not in sgk1-/- MTF. Stimulation of depleted cells with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h) reincreased capacitative Ca(2+) entry in sgk1+/+ MTF, whereas in sgk1-/- cells it remained unchanged. In conclusion, lack of SGK1 does not abrogate Kv channel activity but abolishes regulation of those channels by serum, glucocorticoids and IGF-1, an effect influencing capacitative Ca(2+) entry.
Shumilina
Ekaterina
E
Department of Physiology, University of Tuebingen, Germany.
Lampert
Angelika
A
Lupescu
Adrian
A
Myssina
Svetlana
S
Strutz-Seebohm
Nathalie
N
Henke
Guido
G
Grahammer
Florian
F
Wulff
Peer
P
Kuhl
Dietmar
D
Lang
Florian
F
D000818:Animals; D001798:Blood Proteins / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D005938:Glucocorticoids / Q000494:pharmacology; D017874:Immediate-Early Proteins; D008168:Lung / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D051637:Shaker Superfamily of Potassium Channels; D051670:Shaw Potassium Channels; D013623:Tail / Q000166:cytology; D000099184:Serum-Glucocorticoid Regulated Kinases
D001798:Blood Proteins; D002118:Calcium; D005938:Glucocorticoids; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D051637:Shaker Superfamily of Potassium Channels; D051670:Shaw Potassium Channels; D000099184:Serum-Glucocorticoid Regulated Kinases