Protons and calcium alter gating of the hyperpolarization-activated cation current (I(h)) in rod photoreceptors

We investigated the effects of protons and calcium ions on the voltage-dependent gating of the hyperpolarization-activated, nonselective cation channel current, I(h), in rod photoreceptors. I(h) is a cesium-sensitive current responsible for the peak-plateau sag during the rod response to bright light. The voltage dependence of I(h) activation shifted about 5 mV per pH unit, with external acidification producing positive shifts and alkalinization producing negative shifts. Increasing external [Ca(2+)] from 3 to 20 mM resulted in a large (approximately 17 mV) positive shift in I(h) activation. External [Ca(2+)] (20 mM) blocked pH-induced shifts in activation. Cytoplasmic acidification produced by 25 mM sodium acetate led to a negative shift in inactivation (-9 mV) and internal alkalinization produced with 20 mM ammonium chloride resulted in a positive shift (+6 mV). Surface charge binding and screening theory (Gouy-Chapman-Stern) accounted for the observed shifts in I(h) activation, with the best fit achieved when protons and calcium ions were assumed to bind to distinct sites on the membrane. Since light induces changes in the retinal ionic environment, these results permit us to gauge the degree to which rod light responses could be modified via alterations in I(h) activation.     

NADPH binding to beta-subunit regulates inactivation of voltage-gated K(+) channels

Ancillary beta-subunits regulate the voltage-dependence and the kinetics of Kv currents. The Kvbeta proteins bind pyridine nucleotides with high affinity but the role of cofactor binding in regulating Kv currents remains unclear. We found that recombinant rat Kvbeta 1.3 binds NADPH (K(d)=1.8+/-0.02 microM) and NADP(+) (K(d)=5.5+/-0.9 microM). Site-specific modifications at Tyr-307 and Arg-316 decreased NADPH binding; whereas, K(d) NADPH was unaffected by the R241L mutation. COS-7 cells transfected with Kv1.5 cDNA displayed non-inactivating currents. Co-transfection with Kvbeta1.3 accelerated Kv activation and inactivation and induced a hyperpolarizing shift in voltage-dependence of activation. Kvbeta-mediated inactivation of Kv currents was prevented by the Y307F and R316E mutations but not by the R241L substitution. Additionally, the R316E mutation weakened Kvalpha-beta interaction. Inactivation of Kv currents by Kvbeta:R316E was restored when excess NADPH was included in the patch pipette. These observations suggest that NADPH binding is essential for optimal interaction between Kvalpha and beta subunits and for Kvbeta-induced inactivation of Kv currents.     

Modulation of Kv1.5 potassium channel gating by extracellular zinc.

Zinc ions are known to induce a variable depolarizing shift of the ionic current half-activation potential and substantially slow the activation kinetics of most K(+) channels. In Kv1.5, Zn(2+) also reduces ionic current, and this is relieved by increasing the external K(+) or Cs(+) concentration. Here we have investigated the actions of Zn(2+) on the gating currents of Kv1.5 channels expressed in HEK cells. Zn(2+) shifted the midpoint of the charge-voltage (Q-V) curve substantially more (approximately 2 times) than it shifted the V(1/2) of the g-V curve, and this amounted to +60 mV at 1 mM Zn(2+). Both Q1 and Q2 activation charge components were similarly affected by Zn(2+), which indicated free access of Zn(2+) to channel closed states. The maximal charge movement was also reduced by 1 mM Zn(2+) by approximately 15%, from 1.6 +/- 0.5 to 1.4 +/- 0.47 pC (n = 4). Addition of external K(+) or Cs(+), which relieved the Zn(2+)-induced ionic current reduction, decreased the extent of the Zn(2+)-induced Q-V shift. In 135 mM extracellular Cs(+), 200 microM Zn(2+) reduced ionic current by only 8 +/- 1%, compared with 71% reduction in 0 mM extracellular Cs(+), and caused a comparable shift in both the g-V and Q-V relations (17.9 +/- 0.6 mV vs. 20.8 +/- 2.1 mV, n = 6). Our results confirm the presence of two independent binding sites involved in the Zn(2+) actions. Whereas binding to one site accounts for reduction of current and binding to the other site accounts for the gating shift in ionic current recordings, both sites contribute to the Zn(2+)-induced Q-V shift.     

Single channel analysis reveals different modes of Kv1.5 gating behavior regulated by changes of external pH

In the voltage-gated potassium channel Kv1.5, extracellular acidification decreases the peak macroscopic conductance and accelerates slow inactivation. To better understand the mechanistic basis for these two effects, we recorded unitary currents of Kv1.5 expressed in a mouse cell line (ltk-) using the voltage clamp technique both in cell-attached and excised outside-out patches. Single channel current amplitude at 100 mV (1.7 +/- 0.2 pA at pH 7.4, 1.7 +/- 0.2 pA at pH 6.4) and the single channel conductance between 0 and 100 mV (11.8 +/- 0.6 pS at pH 7.4 and 11.3 +/- 0.8 pS at pH 6.4) did not change significantly with pH. External acidification significantly decreased the number of active sweeps, and this reduction in channel availability accounted for most of the reduction of the peak macroscopic current. The results of runs analyses suggested the null sweeps occur in clusters, and the rate constants for the transition between clusters of null and active sweeps at pH 6.4 were slow (0.12 and 0.18 s(-1), to and from the active clusters, respectively). We propose that low pH facilitates a shift from an available mode (mode A) into an unavailable mode of gating (mode U). In addition to promoting mode U gating, external acidification accelerates depolarization-induced inactivation, which is manifest at the single channel level as a reduction of the mean burst length and an apparent increase of the interburst interval. These effects of external acidification, which are thought to reflect the protonation of a histidine residue in the turret (H-463), point to an important role for the turret in the regulation of channel availability and inactivation.     

Regulation of a mammalian Shaker-related potassium channel, hKv1.5, by extracellular potassium and pH

Using the whole-cell recording mode of the patch-clamp technique we studied the effects of removal of extracellular potassium, [K(+)](o), on a mammalian Shaker-related K(+) channel, hKv1.5. In the absence of [K(+)](o), current through hKv1.5 was similar to currents obtained in the presence of 4.5 mM [K(+)](o). This observation was not expected as earlier results had suggested that either positively charged residues or the presence of a nitrogen-containing residue at the external TEA(+) binding site (R487 in hKv1.5) caused current loss upon removal of [K(+)](o). However, the current loss in hKv1.5 was observed when the extracellular pH, pH(o), was reduced from 7.4 to 6.0, a behavior similar to that observed previously for current through mKv1.3 with a histidine at the equivalent position (H404). These observations suggested that the charge at R487 in hKv1.5 channels was influenced by other amino acids in the vicinity. Replacement of a histidine at position 463 in hKv1.5 by glycine confirmed this hypothesis making this H463G mutant channel sensitive to removal of [K(+)](o) even at pH(o) 7.4. We conclude that the protonation of H463 at pH 7.4 might induce a pK(a) shift of R487 that influences the effective charge at this position leading to a not fully protonated arginine. Furthermore, we assume that the charge at position 487 in hKv1.5 can directly or indirectly disturb the occupation of a K(+) binding site within the channel pore possibly by electrostatic interaction. This in turn might interfere with the concerted transition of K(+) ions resulting in a loss of K(+) conduction.     

The electrically silent Kv6.4 subunit confers hyperpolarized gating charge movement in Kv2.1/Kv6.4 heterotetrameric channels

The voltage-gated K(+) (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ~40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K(+) equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits.     

Association of Kv1.5 and Kv1.3 contributes to the major voltage-dependent K+ channel in macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.     

External pH modulates EAG superfamily K+ channels through EAG-specific acidic residues in the voltage sensor

The Ether-a-go-go (EAG) superfamily of voltage-gated K⁺ channels consists of three functionally distinct gene families (Eag, Elk, and Erg) encoding a diverse set of low-threshold K⁺ currents that regulate excitability in neurons and muscle. Previous studies indicate that external acidification inhibits activation of three EAG superfamily K⁺ channels, Kv10.1 (Eag1), Kv11.1 (Erg1), and Kv12.1 (Elk1). We show here that Kv10.2, Kv12.2, and Kv12.3 are similarly inhibited by external protons, suggesting that high sensitivity to physiological pH changes is a general property of EAG superfamily channels. External acidification depolarizes the conductance–voltage (GV) curves of these channels, reducing low threshold activation. We explored the mechanism of this high pH sensitivity in Kv12.1, Kv10.2, and Kv11.1. We first examined the role of acidic voltage sensor residues that mediate divalent cation block of voltage activation in EAG superfamily channels because protons reduce the sensitivity of Kv12.1 to Zn²⁺. Low pH similarly reduces Mg²⁺ sensitivity of Kv10.1, and we found that the pH sensitivity of Kv11.1 was greatly attenuated at 1 mM Ca²⁺. Individual neutralizations of a pair of EAG-specific acidic residues that have previously been implicated in divalent block of diverse EAG superfamily channels greatly reduced the pH response in Kv12.1, Kv10.2, and Kv11.1. Our results therefore suggest a common mechanism for pH-sensitive voltage activation in EAG superfamily channels. The EAG-specific acidic residues may form the proton-binding site or alternatively are required to hold the voltage sensor in a pH-sensitive conformation. The high pH sensitivity of EAG superfamily channels suggests that they could contribute to pH-sensitive K⁺ currents observed in vivo.     

pH-dependent inhibition of voltage-gated H(+) currents in rat alveolar epithelial cells by Zn(2+) and other divalent cations

Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl(2) reduced the H(+) current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H(+) current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn(2+) were inconsistent with classical voltage-dependent block in which Zn(2+) binds within the membrane voltage field. Instead, Zn(2+) binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn(2+) were strongly pH(o) dependent but were insensitive to pH(i), suggesting that protons and Zn(2+) compete for external sites on H(+) channels. The apparent potency of Zn(2+) in slowing activation was approximately 10x greater at pH(o) 7 than at pH(o) 6, and approximately 100x greater at pH(o) 6 than at pH(o) 5. The pH(o) dependence suggests that Zn(2+), not ZnOH(+), is the active species. Evidently, the Zn(2+) receptor is formed by multiple groups, protonation of any of which inhibits Zn(2+) binding. The external receptor bound H(+) and Zn(2+) with pK(a) 6.2-6.6 and pK(M) 6.5, as described by several models. Zn(2+) effects on the proton chord conductance-voltage (g(H)-V) relationship indicated higher affinities, pK(a) 7 and pK(M) 8. CdCl(2) had similar effects as ZnCl(2) and competed with H(+), but had lower affinity. Zn(2+) applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H(+) currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn(2+) receptor may be the same modulatory protonation site(s) at which pH(o) regulates H(+) channel gating.     

Isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations. Targeting of Kv1.5 to caveolae

The precise subcellular localization of ion channels is often necessary to ensure rapid and efficient integration of both intracellular and extracellular signaling events. Recently, we have identified lipid raft association as a novel mechanism for the subcellular sorting of specific voltage-gated K(+) channels to regions of the membrane rich in signaling complexes. Here, we demonstrate isoform-specific targeting of voltage-gated K(+) (Kv) channels to distinct lipid raft populations with the finding that Kv1.5 specifically targets to caveolae. Multiple lines of evidence indicate that Kv1.5 and Kv2.1 exist in distinct raft domains: 1) channel/raft association shows differential sensitivity to increasing concentrations of Triton X-100; 2) unlike Kv2.1, Kv1.5 colocalizes with caveolin on the cell surface and redistributes with caveolin following microtubule disruption; and 3) immunoisolation of caveolae copurifies Kv1.5 channel. Both depletion of cellular cholesterol and inhibition of sphingolipid synthesis alter Kv1.5 channel function by inducing a hyperpolarizing shift in the voltage dependence of activation and inactivation. The differential targeting of Kv channel subtypes to caveolar and noncaveolar rafts within a single membrane represents a unique mechanism of compartmentalization, which may permit isoform-specific modulation of K(+) channel function.     

Coupling of voltage sensing to channel opening reflects intrasubunit interactions in kv channels

Voltage-gated K(+) channels play a central role in the modulation of excitability. In these channels, the voltage-dependent movement of the voltage sensor (primarily S4) is coupled to the (S6) gate that opens the permeation pathway. Because of the tetrameric structure, such coupling could occur within each subunit or between adjacent subunits. To discriminate between these possibilities, we analyzed various combinations of a S4 mutation (R401N) and a S6 mutation (P511G) in hKv1.5, incorporated into tandem constructs to constrain subunit stoichiometry. R401N shifted the voltage dependence of activation to negative potentials while P511G did the opposite. When both mutations were introduced in the same alpha-subunit of the tandem, the positive shift of P511G was compensated by the negative shift of R401N. With each mutation in a separate subunit of a tandem, this compensation did not occur. This suggests that for Kv channels, the coupling between voltage sensing and gating reflects primarily an intrasubunit interaction.     

Distinct mechanisms of block of Kv1.5 channels by tertiary and quaternary amine clofilium compounds

The quaternary ammonium compound clofilium and its tertiary amine derivative LY97241 were used to analyze mechanisms of block in a voltage-gated potassium channel. Wild-type and mutant Kv1.5 channels expressed in Xenopus oocytes were recorded by two-electrode voltage clamp. Open-channel block to 20% of the control current amplitude was induced reversibly by 50 microM clofilium or 200 microM LY97241, and was seen as an acceleration of the macroscopic current decay. Although blockers remained present after application, channels recovered from block during each interpulse interval. The optimum voltage for recovery (-45 mV at pH 7.3) at the threshold for channel activation indicated that clofilium block and recovery occurred principally through the open channel state. In contrast, LY97241 appeared to exit from the closed state and the open state. In an acid-tolerant Kv1.5 mutant channel (H452Q), external pH was used to titrate LY97241. At low pH, which protonates the LY97241 amine group, recovery from block at hyperpolarized potentials was impaired in a manner similar to that seen with clofilium. Recovery from clofilium block was reduced at negative potentials independent of pH, an effect attributed to trapping of the permanently charged compound within the closed channels.     

Modelling the pH-dependent properties of Kv1 potassium channels

It is known that the pH dependence of conductance for the rat potassium channel Kv1.4 is susbstantially reduced upon mutation of either H508 or K532. These residues lie in the extracellular mouth of the channel pore. We have used continuum electrostatics to investigate their interactions with K(+) sites in the pore. The predicted scale of interactions between H508/K532 and potassium sites is sufficient to significantly alter potassium occupancy and thus channel function. We interpret the effect of K532 mutation as indicating that the pH-dependent effect requires not only an ionisable group with a suitable pK(a) value (i.e. histidine), but also that other charged groups set the potential profile at a threshold level. This hypothesis is examined in the context of pH dependence for other members of the Kv1 family, and may represent a general tool with which to study potassium channels.     

pH determination by pyranine: medium-related artifacts and their correction

Pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) is a water-soluble, membrane-impermeable fluorophore having fluorescent excitation and emission spectra that are highly dependent on medium pH. This combination makes it one of the most commonly used pH-sensitive fluorescent probes to monitor pH and pH changes in biochemical and biophysical research. The pK(a) of this probe is reported to be approximately 7.3, but several studies (including the current one) have shown that this value varies with medium composition. If this is not taken into account, pH determinations based on pyranine may be misleading. We found that in the presence of salts, pK(a) is shifted downward to lower values; therefore, the calculated pH is shifted upward relative to the actual pH as determined by a pH meter. This shift is a consequence of both the type and the concentration of anions and cations that form the salt. Divalent cations cause a larger upward shift in the calculated pH than do monovalent cations. Of all the salts tested, ammonium sulfate has the least effect, and calcium perchlorate has the largest effect, on the pH value calculated by pyranine. Salts are not the only species that affect the pK(a) of pyranine. The presence of the polymer polyethylene glycol (PEG) induces an effect opposite to that of salt (i.e., an upward pK(a) shift), which is expressed as pH being more acidic than that measured by a pH meter. Another nonelectrolyte, dextrose, has no such effect. The effect of both cations and anions can be explained based on their order in the Hofmeister series, whereas the effect of PEG is explained by its high water-binding capacity. Both the ions and PEG change the structure of water and its interaction with pyranine, thereby changing pyranine's apparent pK(a).     

Surface Charges of K channels. Effects of strontium on five cloned channels expressed in Xenopus oocytes

The effects of strontium (Sr2+; 7-50 mM) on five different cloned rat K channels (Kv1.1, Kv1.5, Kv1.6, Kv2.1, and Kv3.4), expressed in oocytes of Xenopus laevis, were investigated with a two-electrode voltage clamp technique. The main effect was a shift of the Gk(V) curve along the potential axis, different in size for the different channels. Kv1.1 was shifted most and Kv3.4 least, 21 and 8 mV, respectively, at 50 mM. The effect was interpreted in terms of screening of fixed surface charges. The estimated charge densities ranged from -0.37 (Kv1.1) to -0.11 (Kv3.4) e nm-2 and showed good correlation with the total net charge of the extracellularly located amino acid residues of the channel as well as with the charge of a specific region (the loop between the S5 segment and the pore forming segment). The estimated surface potentials were found to be linearly related to the activation midpoint potential, suggesting a functional role for the surface charges.     

Apoptosis-Associated Tyrosine Kinase and Neuronal Cell Death

Apoptosis-associated tyrosine kinase (AATYK) is up-regulated by phosphorylation in cultured cerebellar granule neurons (CGN) undergoing apoptosis upon switch to low KCl-containing medium. However, the underlying signaling pathways remain to be fully characterized. When CGN at culture day 7 were switched from 25 mM KCl (K25) to 5 mM (K5) medium, AATYK band migration on SDS–PAGE shifted to a more slowly migrating position expected for the hyperphosphorylated protein. The apoptosis-inducing agent C₂-ceramide also caused a mobility shift of the AATYK protein. Exposing CGN (K25) to L-type voltage-dependent Ca²⁺ channel antagonists shifted the AATYK band to the K5-induced position, while the Ca²⁺ channel activator FPL-64176 had the contrary effect. FK-506, a calcineurin inhibitor caused AATYK hyperphosphorylation under high KCl conditions. CGN death in K5 medium is linked to inhibition of the PI 3-kinase/Akt survival pathway and concomitant activation of the pro-apoptotic downstream target glycogen synthase kinase-3 (GSK-3). GSK-3 inhibitors blocked the K5-induced mobility shift of AATYK. Moreover, CGN cultured from AATYK-deficient mice remained sensitive to death in K5 medium. Thus, AATYK activation may not be a physiologically relevant principal regulatory target of the GSK-3 death pathway in KCl-deprived CGN.     

Different effects of the Ca(2+)-binding protein, KChIP1, on two Kv4 subfamily members, Kv4.1 and Kv4.2.

The Ca(2+)-binding protein, K(+) channel-interacting protein 1 (KChIP1), modulates Kv4 channels. We show here that KChIP1 affects Kv4.1 and Kv4.2 currents differently. KChIP1 slows Kv4.2 inactivation but accelerates the Kv4.1 inactivation time course. Kv4.2 activation is shifted in a hyperpolarizing direction, whereas a depolarizing shift occurs for Kv4.1. On the other hand, KChIP1 increases the current amplitudes and accelerates recovery from inactivation of both currents. An involvement of the Kv4 N-terminus in these differential effects is demonstrated using chimeras of Kv4.2 and Kv4.1. These results reveal a novel interaction of KChIP1 with these two Kv4 members. This represents a mechanism to further increase the functional diversity of K(+) channels.     

Influence of permeating ions on Kv1.5 channel block by nifedipine

Nifedipine can block K(+) currents through Kv1.5 channels in an open-channel manner (32). Replacement of internal and external K(+) with equimolar Rb(+) or Cs(+) reduced the potency of nifedipine block of Kv1.5 from an IC(50) of 7.3 microM (K(+)) to 16.0 microM (Rb(+)) and 26.9 microM (Cs(+)). The voltage dependence of block was unaffected, and a single binding site block model was used to describe block for all three ions. By varying ion species at the intra- and extracellular mouth of the channel and by using a nonconducting W472F-Kv1.5 mutant, we demonstrated that block was conditioned by the ion permeating the pore and, to a lesser extent, by the extracellular ion species alone. In Kv1.5, the outer pore mutations R487V and R487Y reduced nifedipine potency close to that of Kv4.2 and other Kv channels with an equivalent valine. Although changing this residue can affect C-type inactivation of Kv channels, the normalized reduction and time course of currents blocked by nifedipine in 5, 135, and 300 mM extracellular K(+) concentration was the same. Similarly, a mean recovery time constant from nifedipine block of 316 ms was unchanged (332 ms) after 5-s prepulses to allow C-type inactivation. This is consistent with the conclusion that nifedipine block and C-type inactivation in the Kv1.5 channel can coexist but are mediated by distinct mechanisms coordinated by outer pore conformation.     

Protein kinase C mediated pH i -regulation of ROMK1 channels via a phosphatidylinositol-4,5-bisphosphate-dependent mechanism

The protein kinase C (PKC) pathway is important for the regulation of K(+) transport. The renal outer medullar K(+) (ROMK1) channels show an exquisite sensitivity to intracellular protons (pH(i)) (effective pK(a) approximately 6.8) and play a key role in K(+) homeostasis during metabolic acidosis. Our molecular dynamic simulation results suggest that PKC-mediated phosphorylation on Thr-193 may disrupt the PIP(2)-channel interaction via a charge-charge interaction between Thr-193 and Arg-188. Therefore, we investigated the role of PKC and pH(i) in regulation of ROMK1 channel activity using a giant patch clamp with Xenopus oocytes expressing wild-type and mutant ROMK1 channels. ROMK1 channels pre-incubated with the PKC activator phorbol-12-myristate-13-acetate exhibited increased sensitivity to pH(i) (effective pK(a) shifted to pH approximately 7.0). In the presence of GF109203X--a PKC selective inhibitor--the effective pK(a) for inhibition of ROMK1 channels by pH(i) decreased (effective pK(a) shifted to pH approximately 6.5). The pH(i) sensitivity of ROMK1 channels mediated by PKC appeared to be dependent of PIP(2) depletion. The giant patch clamp together with site direct mutagenesis revealed that Thr-193 is the phosphorylation site on PKC that regulates the pH(i) sensitivity of ROMK1 channels. Mutation of PKC-induced phosphorylation sites (T193A) decreases the pH(i) sensitivity and increases the interaction of channel-PIP(2). Taken together, these results provide new insights into the molecular mechanisms underlying the pH(i) gating of ROMK1 channel regulation by PKC.