To study the modulatory effect of extracellular calcium on the whole cell K(+) currents (I(K)) in isolated Deiters cells, the whole cell K(+) currents were recorded when Deiters cells bathed in normal physiological solutions and calcium-free saline, respectively. The electrophysiological characteristics of I(K) currents were then analyzed with the patch clamp technique. Removing extracellular calcium significantly enhanced the amplitude of the I(K) currents, which increased by 70.2% at +50 mV test pulse. The chord conductance, measured at -30 mV test pulse, also significantly increased from (3.31-/+3.08) ns (n=42) in the normal solutions to (10.81-/+6.01) ns (n=42) in the calcium-free solutions, whereas, the zero current potential of the I(K) currents remained unchanged. In calcium-free solutions, the reversal potential of the I(K) currents was shifted to the direction of hyperpolarization, which was very close to the equilibrium K(+) potential based on the Nernst equation. In addition, both the steady state activation curve and the half activation potential, with the averaged value at (-10.13-/+5.64) mV (n=42), were shifted to the negative. However, the tendency for activation (slope conductances) was the same as that in the normal solutions. Interestingly, both the I-V and the G-V functions deduced from the calcium-inhibited K(+) currents in Deiters cells were "S" shape, implying that at least two different kinds of K(+) conductance were involved in this calcium-inhibited K(+) currents. In summary, we hypothesize that there are two mechanisms for this modulation: one is that the I(K) channels in Deiters cells containing a specific calcium sensitive domain, by which extracellular calcium modulates the structure of the K(+) channels and then the I(K) currents; the other is a novel double gated K(+) channel or an ionotropic receptor coupled to K(+) channels or a new subtype of outward K(+) channels. Removing extracellular calcium activates this novel conductance and then modulates the I(K) currents. These results indicate that a decrease in extracellular calcium not only facilitates the efflux of K(+) out of Deiters cells but also accelerates the repolorization by enhancing the I(K) currents, which in turn can effectively buffer the K(+) concentration around the outer hair cells and maintain the resting membrane potential of Deiters cells.
Animals ; Calcium ; physiology ; Cell Separation ; Cochlea ; cytology ; Extracellular Space ; Guinea Pigs ; Membrane Potentials ; physiology ; Patch-Clamp Techniques ; Potassium Channels ; physiology

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.
Animals ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Nanog Homeobox Protein ; genetics ; metabolism ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Patch-Clamp Techniques ; Primary Cell Culture ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Thymosin ; pharmacology