Modulation of the Ca2+-activated large conductance K+ channel by intracellular pH in human renal proximal tubule cells.

The Ca2+-activated and voltage-sensitive large conductance K+ channel (BK channel) with a slope conductance of about 300 pS is present in the surface membrane of cultured human renal proximal tubule epithelial cells (RPTECs). In this study we examined the effects of cytoplasmic pH (pH(i)) on activity and gating kinetics of the BK channel by using the inside-out configuration of the patch-clamp technique. At a constant cytoplasmic Ca(2+) concentration ([Ca2+]i), membrane depolarization raised channel open probability (P(o)), and lowering pH(i) shifted the P(o)-membrane potential (V(m)) relationship to the positive voltage direction. However, the value of the gating charge was not affected by changes in pH(i), suggesting that the effects of pH(i) on P(o) were not due to an alternation of the voltage sensitivity. At constant V(m), lowering pH(i) suppressed the [Ca2+]i-dependent channel activation and shifted the P(o)-[Ca2+]i relationship in the direction of higher [Ca2+]i with a reduction of maximal P(o). Furthermore, both the mean open and mean closed times of the BK channels at pH(i) 6.3 in the presence of 10(-4) M [Ca2+](i) were shorter than those at pH(i) 7.3 in the presence of 10(-5) M [Ca2+]i, even though these two different conditions gave a similar P(o). The data indicate that cytoplasmic H+ suppresses P(o) of the BK channel in RPTECs, which involves the mechanism independent of Ca2+ activation. Our preliminary kinetic analysis also supported this notion.

In vitro 1H-NMR spectroscopic analysis of metabolites in fast- and slow-twitch muscles of young rats.

The lactate (LAC), creatine (CRN), taurine (TAU), anserine (ANS) and carnosine (CAR) content of the masseter muscles (MM), long extensor muscles of digits (EDL) and soleus muscles (SOL) of young rats were determined using in vitro 1H-NMR spectroscopy to assess the significance of CRN, TAU, ANS and CAR in these muscles. The muscles of Wistar rats at the ages of 6, 12 and 18 weeks were dissected after decapitation and used for the metabolite analyses. The LAC and CAR content of all muscle groups showed no age dependence. The CRN content was increased age-dependently in MM but not in EDL or SOL. The LAC and CRN content was higher in MM and EDL (fast-twitch) than in SOL (slow-twitch) (P<0.01-0.001 at 18 weeks). A significant positive correlation existed between the LAC and CRN content (P<0.00001, r=0.80), suggesting that the CRN content reflects the capacity of the anaerobic glycolysis of the individual muscles. The TAU content was higher in SOL and MM than in EDL (P<0.05) and showed an approximately 1.5-fold increase with age in all three muscle groups. The ANS content was higher in EDL than in SOL and MM (P<0.001), and showed an approximately threefold increase with age in all three muscle groups. The ANS content positively correlated with the LAC content (P<0.001, r=0.41), and the chemical shift of the imidazole proton in ANS showed a correlation with the LAC content (P<0.0001, r>0.76), indicating that ANS would buffer the pH change produced by LAC. These results suggest that 1H-NMR spectroscopy would provide an adjunct method of assessing the muscle types and their development.

Protein kinase G activates inwardly rectifying K(+) channel in cultured human proximal tubule cells.

An ATP-regulated inwardly rectifying K(+) channel, whose activity is enhanced by PKA, is present in the plasma membrane of cultured human proximal tubule cells. In this study, we investigated the effects of PKG on this K(+) channel, using the patch-clamp technique. In cell-attached patches, bath application of a membrane-permeant cGMP analog, 8-bromoguanosine 3',5'-monophosphate (8-BrcGMP; 100 microM), stimulated channel activity, whereas application of a PKG-specific inhibitor, KT-5823 (1 microM), reduced the activity. Channel activation induced by 8-BrcGMP was observed even in the presence of a PKA-specific inhibitor, KT-5720 (500 nM), which was abolished by KT-5823. Direct effects of cGMP and PKG were examined with inside-out patches in the presence of 1 mM MgATP. Although cytoplasmic cGMP (100 microM) alone had little effect on channel activity, subsequent addition of PKG (500 U/ml) enhanced it. Furthermore, bath application of atrial natriuretic peptide (ANP; 20 nM) in cell-attached patches stimulated channel activity, which was blocked by KT-5823. In conclusion, cGMP/PKG-dependent processes participate in activating the ATP-regulated K(+) channel and producing the stimulatory effect of ANP on channel activity.