Shift In pH Dependence Research Articles

PH dependence of the Slc4a11-mediated H+ conductance is influenced by intracellular lysine residues and modified by disease-linked mutations.

SLC4A11 is the only member of the SLC4 family that transports protons rather than bicarbonate. SLC4A11 is expressed in corneal endothelial cells, and its mutation causes corneal endothelial dystrophy, although the mechanism of pathogenesis is unknown. We previously demonstrated that the magnitude of the H+ conductance (Gm) mediated by SLC4A11 is increased by rises in intracellular as well as extracellular pH (pHi and pHe). To better understand this feature and whether it is altered in disease, we studied the pH dependence of wild-type and mutant mouse Slc4a11 expressed in Xenopus oocytes. Using voltage-clamp circuitry in conjunction with a H+-selective microelectrode and a microinjector loaded with NaHCO3, we caused incremental rises in oocyte pHi and measured the effect on Gm. We find that the rise of Gm has a steeper pHi dependence at pHe =8.50 than at pHe =7.50. Data gathered at pHe =8.50 can be fit to the Hill equation enabling the calculation of a pK value that reports pHi dependence. We find that mutation of lysine residues that are close to the first transmembrane span (TM1) causes an alkaline shift in pK. Furthermore, two corneal-dystrophy-causing mutations close to the extracellular end of TM1, E399K and T401K (E368K and T370K in mouse), cause an acidic shift in pK, while a third mutation in the fourth intracellular loop, R804H (R774H in mouse), causes an alkaline shift in pK. This is the first description of determinants of SLC4A11 pH dependence and the first indication that a shift in pH dependence could modify disease expressivity in some cases of corneal dystrophy.

Abstract 17301: Functional Characterization of KCNK3 Mutants Associated With Pulmonary Arterial Hypertension Under Physiologically Relevant Heterozygous Conditions

KCNK3 encodes a two-pore domain K+ channel, TASK-1, which is inhibited by extracellular acidity and hypoxia. Expressed in a variety of tissues, including human pulmonary artery smooth muscle cells (hPASMCs), the central nervous system, pancreas, and adrenal glands, TASK-1 contributes to the resting membrane potential of cells in which it is expressed. Recently, our group reported mutations in KCNK3 underlying idiopathic pulmonary arterial hypertension (PAH), resulting from loss of TASK-1 function, partially pharmacologically rescuable with ONO-RS-082. TASK-1 dimerizes in vivo, forming functional channels with another TASK-1 subunit or with the related TASK-3 channel. TASK-1 and TASK-3 often are expressed in the same cells, although it has been reported that TASK-1 alone is expressed in the lung. Our initial study examined mutant and wildtype (WT) homodimeric TASK-1 channels expressed heterologously in COS-7 cells. Here we further characterize PAH-linked TASK-1 mutations in physiologically relevant heterozygous conditions in COS-7 and hPASMC cell lines. We engineered heterodimeric channels consisting of one mutant and one WT subunit; compared this with co-expression of mutant and WT channels; and measured channel activity with whole cell patch clamp procedures. We found a mutation specific impact of heterozygosity on channel activity. One mutation, V221L, produces a shift in pH dependence accounting for loss of function at physiological pH 7.4, partially rescued by dimerization with a WT subunit, while another, G203D, produces near complete loss of function as a homo- or hetero-dimer. The presence of TASK-3 results in greater rescue of V221L TASK-1 activity at pH 7.4 than does WT TASK-1. Additionally, under current clamp we found that ONO-RS-082 hyperpolarizes the membrane potential in hPASMCs expressing WT or V221L TASK-1, reversible by selective block of TASK-1 with ML365. Together, our results suggest (a) TASK-1 mutant heterodimers exhibit loss of function with mutation specific severity; (b) TASK-3 may rescue mutant TASK-1 and underlie a tissue specific impact of the TASK-1 mutations observed clinically; and (c) PAH TASK-1 mutants can be pharmacologically modulated in hPASMCs and alter the critically important resting membrane potential.

Functional importance of charged residues within the putative intracellular loops in pH regulation by Na+/ H+exchanger NHE1

The plasma membrane Na+/H+ exchanger 1 is activated in response to various extrinsic factors, and this process is regulated by an intracellular pH-sensing mechanism. To identify the candidate residues responsible for intracellular pH regulation, we analyzed the functional properties of engineered Na+/H+ exchanger 1 mutants with charge-reversal mutations of charged residues located in the intracellular loops. Na+/H+ exchanger 1 mutants with mutations at 11 positions were well expressed in the plasma membrane, but that with E247R was not, suggesting that Glu247 is important for the functional expression of Na+/H+ exchanger 1. Charge-reversal mutations of Glu131 (E131R, E131K) and Arg327 (R327E) resulted in a shift in the intracellular pH dependence of the exchange activity measured by 22Na+ uptake to the acidic side, and it abolished the response to growth factors and a hyperosmotic medium; however, mutations of Asp448 (D448R) and Arg500 (R500E) slightly shifted it to the alkaline side. In E131R, in addition to the change in intracellular pH dependence, the affinities for extracellular Na+, Li+ and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride significantly increased. Furthermore, charge-conserved mutation of E131 (E131D) was found to have no effect, whereas charge neutralization (E131Q) resulted in a slight acidic shift of exchange. These results support the view that the multiple charged residues identified in this study, along with several basic residues reported previously, participate in the regulation of the intracellular pH sensing of Na+/H+ exchanger 1. In addition, Glu131 may also be important for cation transport.

Abnormal pH-sensing of platelet NA +/H + exchanger in patients with cardiac syndrome X

An enhanced activity of Na +/Li + countertransport, studied as a surrogate of Na +/H + exchanger, has been described in red blood cells of patients with cardiac syndrome X. In this study we investigated whether abnormalities in the activity of platelet Na +/H + exchanger (NHE) also existed in syndrome X patients and whether such abnormality was associated with platelet activation. Platelet NHE activity was evaluated in 21 syndrome X patients and 18 controls by measuring the pH recovery in platelets after acid loading and/or thrombin stimulation. The linear correlation existing between the initial intracytoplasmic pH (pH i) values and the maximal velocity of pH recovery allowed to calculate the values of slope and intercept at pH i=6.6 ( I pH6.6) for each individual. Urinary excretion of the major TXB 2 metabolite, 11-dehydro-TXB 2 was measured in 15 syndrome X patients and 15 controls. The acidification-induced NHE activity resulted significantly higher in syndrome X patients compared to controls. Indeed, slope values were 0.75±0.29 and 0.5±0.23 min −1 in patients and controls, respectively ( P=0.01), while I pH6.6 values were 0.24±0.1 and 0.17±0.1 ΔpH/min ( P=0.04). The thrombin-stimulated NHE activity, however, was not different in the two groups and no significant difference in the urinary excretion of 11-dehydro-TXB 2 between patients and controls (median 920 vs. 765 pg/mg creatinine, respectively) ( P=0.32) was also found. Thus our data demonstrate an alkaline shift in pH-dependence of platelet NHE of syndrome X patients. This abnormality does not seem to be associated with increased platelet activation.

Regulation of inward rectifier K+ channels by shift of intracellular pH dependence.

The mechanistic link between mitochondrial metabolism and inward rectifier K+ channel activity was investigated by studying the effects of a mitochondrial inhibitor, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on inward rectifiers of the Kir2 subfamily expressed in Xenopus oocytes, using two-electrode voltage-clamp, patch-clamp, and intracellular pH recording. FCCP inhibited Kir2.2 and Kir2.3 currents and decreased intracellular pH, but the pH change was too small to account for the inhibitory effect by itself. However, pre-incubation of oocytes with imidazole prevented both the pH decrease and the inhibition of Kir2.2 and Kir2.3 currents by FCCP. The pH dependence of Kir2.2 was shifted to higher pH in membrane patches from FCCP-treated oocytes compared to control oocytes. Therefore, the inhibition of Kir2.2 by FCCP may involve a combination of intracellular acidification and a shift in the intracellular pH dependence of these channels. To investigate the sensitivity of heteromeric channels to FCCP, we studied its effect on currents expressed by heteromeric tandem dimer constructs. While Kir2.1 homomeric channels were insensitive to FCCP, both Kir2.1-Kir2.2 and Kir2.1-Kir2.3 heterotetrameric channels were inhibited. These data support the notion that mitochondrial dysfunction causes inhibition of heteromeric inward rectifier K+ channels. The reduction of inward rectifier K+ channel activity observed in heart failure and ischemia may result from the mitochondrial dysfunction that occurs in these conditions.

The Molecular Basis for the pH-activation Mechanism in the Channel-forming Bacterial Colicin E1

The in vitro activity of the channel-forming bacteriocins such as colicin E1 in model membranes requires the specific activation of the protein by an acidic environment in the presence of a membrane potential. Acid activation of the C-terminal domain results in the formation of an insertion-competent intermediate with an enhanced ability to penetrate and perforate cell membranes. We report novel findings of this activation process through the design and study of mutant proteins involving the replacement of conserved Asp residues Asp-408, Asp-410, and Asp-423 within helices 5a and 4 in the colicin E1 channel domain that resulted in enhanced membrane binding, bilayer insertion rates, and ion channel activities at near neutral pH values. This activation process involves the destabilization of a critical salt bridge (Asp-410 and Lys-406) and H-bonds (Asp-408 and Ser-405 main chain; Asp-423 and Lys-420 main chain). The helix-to-coil transition of this motif was identified previously by time-resolved Trp fluorescence measurements (Merrill, A. R., Steer, B. A., Prentice, G. A., Weller, M. J., and Szabo, A. G. (1997) Biochemistry 36, 6874-6884), and here we use this approach to demonstrate that disruption of the helical structure of helices 4 and 5a results in a shift in this equilibrium to favor the coil state. Finally, we show that the essential components of the pH trigger motif are conserved among the channel-forming colicins and that it likely exists within other bacterial proteins and may even have evolved into more sophisticated devices in a number of microbial species.

Mutations of Arg440 and Gly455/Gly456 Oppositely Change pH Sensing of Na+/H+ Exchanger 1

To identify important amino acid residues involved in intracellular pH (pH(i)) sensing of Na(+)/H(+) exchanger 1, we produced single-residue substitution mutants in the region of the exchanger encompassing the putative 11th transmembrane segment (TM11) and its adjacent intracellular (intracellular loop (IL) 5) and extracellular loops (extracellular loop 6). Substitution of Arg(440) in IL5 with other residues except positively charged Lys caused a large shift in pH(i) dependence of (22)Na(+) uptake to an acidic side, whereas substitution of Gly(455) or Gly(456) within the highly conserved glycine-rich sequence of TM11 shifted pH(i) dependence to an alkaline side. The observed alkaline shift was larger with substitution of Gly(455) with residues with increasing sizes, suggesting the involvement of the steric effect. Interestingly, mutation of Arg(440) (R440D) abolished the ATP depletion-induced acidic shift in pH(i) dependence of (22)Na(+) uptake as well as the cytoplasmic alkalinization induced by various extracellular stimuli, whereas with that of Gly(455) (G455Q) these functions were preserved. These mutant exchangers did not alter apparent affinities for extracellular transport substrates Na(+) and H(+) and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride. These results suggest that positive charge at Arg(440) is required for normal pH(i) sensing, whereas mutation-induced perturbation of the TM11 structure may be involved in the effects of Gly mutations. Thus, both Arg(440) in IL5 and Gly residues in the conserved segment of TM11 appear to constitute important elements for proper functioning of the putative "pH(i) sensor" of Na(+)/H(+) exchanger 1.

Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H(+)-sensitive and Ca2+ regulation-defective.

The ubiquitous plasma membrane Na+/H+ exchanger (NHE1) is rapidly activated in response to various extracellular signals. To understand how the intracellular Ca2+ is involved in this activation process, we investigated the effect of Ca2+ ionophore ionomycin on activity of the wild-type or mutant NHE1 expressed in the exchanger-deficient fibroblasts (PS120). In wild-type transfectants, a short (up to 1 min) incubation with ionomycin induced a significant alkaline shift (approximately 0.2 pH unit) in the intracellular pH (pHi) dependence of the rate of 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ uptake, without changes in the cell volume and phosphorylation state of NHE1. Mutations that prevented calmodulin (CaM) binding to a high affinity binding region (region A, amino acids 636-656) rendered NHE1 constitutively active by inducing a similar alkaline shift in pHi dependence of Na+/H+ exchange. These same mutations abolished the ionomycin-induced NHE1 activation. These data suggest that CaM-binding region A functions as an "autoinhibitory domain" and that Ca2+/CaM activates NHE1 by binding to region A and thus abolishing its inhibitory effect. Furthermore, we found that a short stimulation with thrombin and ionomycin had apparently no additive effects on the alkaline shift in the pHi dependence of Na+/H+ exchange and that deletion of region A also abolished such an alkaline shift induced by a short thrombin stimulation. The results strongly suggest that the early thrombin response and the ionomycin response share the same activation mechanism. Based on these data and the results shown in the accompanying paper (Bertrand, B., Wakabayashi, S., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709), we propose that CaM is one of the major "signal transducers" that mediate distinct extracellular signals to the "pHi sensor" of NHE1.

Activation of the Na+/H+ exchanger by phorbol ester and osmotic shock is dependent on the degree of neutrophilic maturation

Activation of neutrophils leading to superoxide production is accompanied by cytoplasmic alkalinization, which results from stimulation of the Na+/H+ exchanger. Since the exchanger undergoes permanent alterations during neutrophilic maturation of HL-60 cells (Costa-Casnellie et al.: Journal of Biological Chemistry 263:11851-11855, 1988), we investigated whether its response to external stimuli such as phorbol esters or osmotic shock also was modified during cell maturation. Mature HL-60 cells produce superoxide in response to active phorbol esters, whereas immature HL-60 cells do not. Stimulation of the exchanger by active phorbol esters (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) was observed in mature neutrophilic HL-60 cells but not in their immature counterparts. Inactive 4-alpha phorbol had no effect in either cell population. Compound H7 inhibited phorbol ester activation by 65%. In mature neutrophilic cells activation of the exchanger by phorbol esters caused two novel changes of its properties: 1) its apparent Km for Na+ transport increased 2-fold; 2) its Vmax increased 2.6-fold. Phorbol esters also caused a shift in pH dependence of activation similar to that induced in other cells. Osmotic shock, a different method known to activate the exchanger of other cells, induced activation in mature neutrophilic cells but not in immature cells. Thus, the response of the exchanger to external stimuli is affected by alterations occurring in association with cell maturation.