Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor

Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A I M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, ³⁵SO₄ was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3 M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3–7.2, to intracellular concentrations of at least 40–200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4–8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.     

A quantitative comparison of the effects of intracellular calcium injection and light adaptation on the photoresponse of Limulus ventral photoreceptors

Calcium ions were iontophoretically injected into ventral photoreceptors of Limulus by passing current between two intracellular pipettes. Changes in sensitivity and photoresponse time course were measured for both light adaptation and Ca++ injection. We found for some photoreceptors that there was no significant difference in the photoresponse time course for desensitization produced by light adaptation or by Ca++ injection. In other photoreceptors, the time delay of photoresponse for Ca++ injection was slightly longer than for light adaptation. The variability of threshold response amplitude and time delay decreases when the photoreceptor is desensitized by either light adaptation or Ca++ injection. The peak amplitude versus log stimulus intensity relationships for controls, light adaptation, and Ca++ injection all could be described very closely by a single template curve shifted along the log intensity axis. A 40- to 50-fold change in sensitivity is associated with a 2-fold change in photoresponse time delay for both light adaptation and Ca++ injection.     

Effects of intracellular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors

The calcium sequestering agent, EGTA, was injected into Limulus ventral photoreceptors. Before injection, the inward membrane current induced by a long stimulus had a large initial transient which declined to a smaller plateau. Iontophoretic injection of EGTA tended to prevent the decline from transient to plateau. Before injection the plateau response was a nonlinear function of light intensity. After EGTA injection the response-intensity curves tended to become linear. Before injection, bright lights lowered the sensitivity as determined with subsequent test flashes. EGTA injection decreased the light-induced changes in sensitivity. Ca-EGTA buffers having different levels of free calcium were pressure-injected into ventral photoreceptors; the higher the level of free calcium, the lower the sensitivity measured after injection. The effects of inotophoretic injection of EGTA were not mimicked by injection or similar amounts of sulfate and the effects of pressure injection of EGTA buffer solutions were not mimicked by injection of similar volumes of pH buffer or mannitol. The data are consistent with the hypothesis that light adaptation is mediated by a rise of the intracellular free calcium concentration.     

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.     

Electrochemical DNA biosensor for the study of ciprofloxacin-DNA interaction

The interaction of ciprofloxacin with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of ciprofloxacin was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current at +0.9 V was used as an indicator for the interaction mechanism in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 1.33+/-0.02 x 10(4) and 1.32+/-0.08 x 10(4) M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak currents was observed in the range of 40-80 microM ciprofloxacin, with a detection limit of 24 microM with r=0.995 and 9 microM with r=0.999 by using constant current potentiometry and differential pulse voltammetry, respectively. Moreover, the influence of sodium and calcium ions was also studied to elucidate the mechanism of ciprofloxacin-DNA interaction at different solution conditions, and this proved to be helpful in understanding the ciprofloxacin-DNA interaction.     

Physiological and morphological identification of photosensitive neurons in the opisthosomal ganglia of Limulus polyphemus

The motor outputs of the isolated opisthosomal ventral nerve cord in Limulus polyphemus are modulated by light. We have identified the photosensitive neurons and examined their physiological and morphological properties using intracellular recording and staining techniques. We found that photosensitive neurons are present in each ganglion of the opisthosomal ventral nerve cord. These neurons often discharged action potentials spontaneously in the dark, and they increased the frequency of this discharge in the light. The mean latency (+/-SD) of the light-induced action potential was 2.2 +/- 1.1 s. Cells responded in a graded fashion over a 2-log unit of light intensity. The peak spectral sensitivity was 425 nm or lower. The Lucifer-yellow-labeled photosensitive neurons had oval somata with mean (+/-SD) diameters of 102 +/- 3 microm (long axis) and 75 +/- 5 microm (short axis), and extended their axons to the contralateral region of the ventral nerve cord. The soma had no dendrites, and the axon had thin branches.     

Inositol 1,4,5-trisphosphate-induced calcium release is necessary for generating the entire light response of limulus ventral photoreceptors

The experiments reported here were designed to answer the question of whether inositol 1,4,5-trisphosphate (IP3)-induced calcium release is necessary for generating the entire light response of Limulus ventral photoreceptors. For this purpose the membrane-permeable IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2APB) (Maruyama, T., T. Kanaji, S. Nakade, T. Kanno, and K. Mikoshiba. 1997. J. Biochem. (Tokyo). 122:498-505) was used. Previously, 2APB was found to inhibit the light activated current of Limulus ventral photoreceptors and reversibly inhibit both light and IP3 induced calcium release as well as the current activated by pressure injection of calcium into the light sensitive lobe of the photoreceptor (Wang, Y., M. Deshpande, and R. Payne. 2002. Cell Calcium. 32:209). In this study 2APB was found to inhibit the response to a flash of light at all light intensities and to inhibit the entire light response to a step of light, that is, both the initial transient and the steady-state components of the response to a step of light were inhibited. The light response in cells injected with the calcium buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was reversibly inhibited by 2APB, indicating that these light responses result from IP3-mediated calcium release giving rise to an increase in Cai. The light response obtained from cells after treatment with 100 microM cyclopiazonic acid (CPA), which acts to empty intracellular calcium stores, was reversibly inhibited by 2APB, indicating that the light response after CPA treatment results from IP3-mediated calcium release and a consequent rise in Cai. Together these findings imply that IP3-induced calcium release is necessary for generating the entire light response of Limulus ventral photoreceptors.     

The spectral sensitivity of single units in the nucleus rotundus of pigeon, Columba livia

Responses to diffuse monochromatic light were recorded from single units in the diencephalon of pigeon. Units were both excited and inhibited by light stimulation. Intensity-response functions based on latency measures to the first spike after stimulation were used to generate action spectra. One class of spectral sensitivity functions presumably from rods, showed peak sensitivities near 500 nm: these functions were unaffected by changing criterion values used to generate the functions. A second class of cone functions showed multiple peak sensitivities at 540 nm and 600–620 nm. These units shifted their peak sensitivities with a change in criterion values. Unit response types tended to be localized differentially in the nucleus rotundus. Excitatory units were located in the dorsal half of the nucleus, while inhibitory units were located in the ventral half, with a few exceptions. An attempt was made to integrate the present findings with previous behavioral, electrophysiological, photochemical, and anatomical data in the pigeon.     

Ischemia alters the electrical activity of pacemaker cells isolated from the rabbit sinoatrial node

The purpose of this study was to investigate the mechanisms responsible for ischemia-induced changes in spontaneous electrical activity. An ischemic-like Tyrode solution (pH 6.6) reversibly depolarized the maximum diastolic potential (MDP) and reduced the action potential (AP) overshoot (OS). We used SNARF-1, which is an indicator of intracellular pH (pH(i)), and perforated-patch techniques to test the hypothesis that acidosis caused these effects. Acidic but otherwise normal Tyrode solution (pH 6.8) produced similar effects. Basic Tyrode solution (pH 8.5) hyperpolarized the MDP, shortened the AP, and slowed the firing rate. In the presence of "ischemic" Tyrode solution, hyperpolarizing current restored the MDP and OS to control values. HOE-642, an inhibitor of Na/H exchange, did not alter pH(i) or electrical activity and did not prevent the effects of ischemic Tyrode solution or recovery after washout. Time-independent net inward current but not hyperpolarization-activated inward current was enhanced by ischemic Tyrode solution or by 30 microM BaCl(2), a selective blocker of inward-rectifying K currents at this concentration. The results suggest that 1) acidosis was responsible for the ischemia-induced effects but Na/H exchange was not involved, 2) the OS was reduced because of depolarization-induced inactivation of inward currents that generate the AP upstroke, and 3) reduction of an inward-rectifying outward K current contributed to the depolarization.     

Cardiodepressive effect of platelet activating factor

The cardiodepressive effect of PAF has been studied on the electrical and mechanical activities of isolated auricles of guinea pig. Intracellular resting potential, action potential (AP) and isometric contractions elicited by electrical stimulation (0.5 Hz) were measured. PAF (10(-7) M) induced negative inotropic effect, which reached its peak after 5 min with 23.5 +/- 6.6% in respect to prechallenge values (n = 8). After 20 min negative inotropic effect relaxed to 39.6 +/- 8.8%. 1 min after the beginning of washing in Tyrode solution, positive inotropic effect of PAF was evident, that reached its peak (217 +/- 49.5%) after 2 min, decayed after 5-10 min to normal values. PAF did not modify the resting membrane potential, produced a decrease in the amplitude and Vmax of the upstroke AP, shortened the AP duration. Ca-AP and contractions, elicited in partially depolarized myocardium were decreased by PAF (10(-7) M). PAF-produce the change of the AP and the negative effect on auricle contractile force was inhibited in muscles pretreated with 3mM 4 aminopyridine. Histamine (10(-4) M) was also capable of neutralizing the depressant effect of PAF. The obtained results suggested that PAF effects on the membrane of cardiac cells could be related to a change in Ca and K conductance.     

A novel approach for the selective determination of tryptophan in blood serum in the presence of tyrosine based on the electrochemical reduction of oxidation product of tryptophan formed in situ on graphite electrode

In this study, a novel method was proposed for the selective determination of tryptophan (TRP) in blood serum in the presence of tyrosine. This method is based on the electrochemical reduction of 2-amino-3-(5-oxo-3,5-dihydro-2H-indol-3-yl)-propionic acid (5-O-3,5DH-TRP) formed by the oxidation of TRP on the electrochemically treated pencil graphite (ETPG) electrode surface at a suitable potential value. The parameters affecting the TRP determination were deeply investigated. The optimal pH value was determined as 3. The highest reduction current intensity was obtained at the accumulation potential and time values of +0.95 V and 120 s, respectively. The reduction peak current values of 5-O-3,5DH-TRP versus TRP concentration at the ETPG electrode showed linearity in the range from 0.5 μM to 50.0 μM (R(2)=0.9962) with a detection limit of 0.05 μM (S/N=3). The reduction peak intensity of 5-O-3,5DH-TRP on the ETPG electrode showed no significant change in the presence of different interfering substances. The analytical application of the proposed novel method was successfully tested by using human blood serum samples.     

Relationship between light sensitivity and intracellular free Ca concentration in Limulus ventral photoreceptors. A quantitative study using Ca-selective microelectrodes

The possible role of Ca ions in mediating the drop in sensitivity associated with light adaptation in Limulus ventral photoreceptors was assessed by simultaneously measuring the sensitivity to light and the intracellular free Ca concentration (Cai); the latter was measured by using Ca-selective microelectrodes. In dark-adapted photoreceptors, the mean resting Cai was 3.5 +/- 2.5 microM SD (n = 31). No correlation was found between resting Cai and absolute sensitivity from cell to cell. Typically, photoreceptors are not uniformly sensitive to light; the Cai rise evoked by uniform illumination was 20-40 times larger and faster in the most sensitive region of the cell (the rhabdomeral lobe) than it was away from it. In response to a brief flash, the Cai rise was barely detectable when 10(2) photons were absorbed, and it was saturated when approximately 10(5) photons were absorbed. During maintained illumination, starting near the threshold of light adaptation, steady Cai increases were associated with steady desensitizations over several log units of light intensity: a 100-fold desensitization was associated with a 2.5-fold increase in Cai. After a bright flash, sensitivity and Cai recovered with different time courses: the cell was still desensitized by approximately 0.5 log units when Cai had already recovered to the prestimulus level, which suggests that under those conditions Cai is not the rate-limiting step of dark adaptation. Ionophoretic injection of EGTA markedly decreased the light-induced Cai rise and increased the time to peak of the light response, but did not alter the resting Cai, which suggests that the time to peak is affected by a change in the capacity to bind Ca2+ and not by resting Cai. Lowering the extracellular Ca2+ concentration (Cao) first decreased Cai and increased sensitivity. Longer exposure to low Cao resulted in a further decrease of Cai but decreased rather than increased sensitivity, which suggests that under certain conditions it is possible to uncouple Cai and sensitivity.     

Studies on the state of tyrosyl residues in a ribonuclease from seminal vesicles.

In order to study the state of tyrosyl residues in a ribouuclease from bovine semina vesicles [EC 3.1.4.22, RNase Vs1] several lines of experiments were carried out. Spectrophotometric titration of RNase Vs1 indicated that two out of 8 tyrosine residues were titrated very easily and their apparent pKa values were about 9.8. Next, about 4 residues were titrated at pH up to 13.5. The remaining 2 residues were titrated time-dependently at pH 13.5. In 8 M urea, about 6 tyrosine residues were titrated with apparent pK4 values of about 11.2 and about 2 residues were titrated time-dependently at pH 13.5. Acetylation of RNase Vs1 with N-acetylimidazole was studied at pH 7.5. In aqueous solution, about 1.1-3.5 tyrosine residues were acetylated, depending on the experimental conditions, and in 8 M urea, 5.3 tyrosine residues were modified. RNase Vs1 was nitrated with tetranitromethane at pH 7.5. In aqueous solution, about 2.5 tyrosine residues were nitrated very easily; the enzymatic activity of the modified enzymes was 130-200% of that of the native enzyme. In 8 M urea, the reactivity of the tyrosine residues increased and about 4-5.5 residues were modified. The results of chemical modification and spectrophotometric titration indicated that about two tyrosine residues in RNase Vs1 were exposed to the solvent and were more reactive to various reagents, and 3-4 tyrosine residues were less reactive. The final 2 residues were not accessible to the reagent even in the presence of urea, but were titraten at pH 13.5. The solvent perturbation difference spectrum using ethylene glycol as a perturbant indicated that about 4 tyrosine residues were perturbed. When the pH of the enzyme solution was changed from 7.0 to 1.0, the change in optical density of RNase Vs1 due to denaturation blue shift was about 1,600 at 287nm. The optical density change at 287 nm of native RNase Vs1 on exposure to 8 M urea and 6 M guanidine-HCl indicated that the environments of 2-3 and 4 tyrosine residues were changed by the addition of the denaturants, urea and guanidine-HCl, respectively. In RNase Vs1 having about four nitrotyrosine residues, the two most inaccessible tyrosine residues remained resistant to titration with alkali. On adding nucleotide, nitrated RNase Vs1 gave a difference spectrum in the ultraviolet region but not in 320-460 nm region, where nitrotyrosine residues absorb light. This may indicate that tyrosine residues located relatively near the surface of the molecule are not perturbed directly by nucleotide binding.     

Photoreaction of bacteriorhodopsin at high pH: origins of the slow decay component of M.

The absorption spectrum of light-adapted purple membrane in 3 M KCl is dependent on temperature even in the room temperature region. Temperature-induced difference spectra at various pH values suggested that the trans isomer of bacteriorhodopsin, bR570, is in thermal and/or photodynamic equilibrium with several different conformers. The major second conformer occurring at neutral pH had the same spectroscopic properties as the 13-cis isomer, and its content at 35 degrees C was estimated to be more than 20%. Heterogeneity in the protein conformation became more significant above pH8, where temperature-induced difference spectra exhibited a negative peak at 580 nm and a positive peak at 296 nm. This absorption change is very similar to that observed upon the formation of the N intermediate, suggesting that an N-like conformer occurs at high pH and temperature. A significant temperature dependence was also seen in the M decay kinetics at high pH, which were described by two decay components; i.e., the fast decaying M (Mf) was predominant at low temperature, but the amplitude of the slow component (M(s)) increased with increasing temperature. It is suggested that M(s) is generated upon excitation of the N-like conformer, in which the residue (Asp-96) usually acting as a proton donor to the Schiff base is deprotonated. The N-like conformer could be N itself, because M(s) was enhanced when N was accumulated by background light. A strong correlation between the amplitude of M(s) and the concentration of N was also revealed by the accumulation kinetics of Mf, M(s), and N after the onset of continuous actinic light.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a subsurface area in the ventral medulla sensitive to local changes in PCO2.

The exact location of the central respiratory chemoreceptors sensitive to changes in PCO2 has not yet been determined. To avoid the confounding effects of the cerebral circulation, we used the in vitro brain stem-spinal cord of neonatal rats (1-5 days old) to identify areas within 500 microns of the ventral surface of the medulla where changes in PCO2 evoked a sudden increase in the rate of respiratory neural activity. The preparation was superfused with mock cerebrospinal fluid (CSF) while maintained at constant temperature (26 +/- 1 degrees C) and pH (7.34). Respiratory frequency increased linearly with decreases in superfusate pH (r2 = 0.92, P less than 0.001), indicating that the respiratory circuitry for the detection of CO2 and stimulation of breathing was intact in this preparation. The search for central chemoreceptors was performed with a specially designed micropipette that allowed microejection of 2-10 nl of mock CSF equilibrated with different CO2-O2 gas mixtures. The pipette was advanced in 50- to 100-microns steps by use of a microdrive to a maximum depth of 500 microns from the surface of the ventral medulla. Depending on the location of the micropipette, ejection of CO2-acidified mock CSF at depths of 100-350 microns below the ventral surface of the medulla stimulated neural respiratory output. Using this response as an indication of the location of central respiratory chemoreceptors, we found that chemoreceptive elements were located in a column in the ventromedial medulla extending from the hypoglossal rootlets caudally to an area 0.75 mm caudal to VI nerve in the rostral medulla.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular pH modifies mitochondrial control of capacitative calcium entry in Jurkat cells

It was found that a collapse of the mitochondrial calcium buffering caused by the protonophoric uncoupler CCCP, antimycin A plus oligomycin, or the inhibitor of the mitochondrial Ca2+/Na+ exchanger led to a strong inhibition of thapsigargin-induced capacitative Ca2+ entry (CCE) into Jurkat cells suspended in a medium at pH 7.2. The effect of these inhibitors was markedly less significant at higher extracellular pH. Moreover, dysfunction of the mitochondrial calcium handling greatly decreased CCE sensitivity to extracellular Ca2+ when the pH of extracellular solution was 7.2 (apparent Kd toward extracellular Ca2+ rose from 2.3 +/- 0.6 mm in control cells to 11.0 +/- 1.7 mM in CCCP-treated cells) as compared with pH 7.8 (apparent Kd toward extracellular Ca2+ increased from 1.3 +/- 0.4 mM in control cells to 2.4 +/- 0.4 mM in uncoupler-treated cells). Changes in intracellular pH triggered by methylamine did not influence Ca2+ influx. This suggests that, in Jurkat cells, store-operated calcium channels sense extracellular pH change as a parameter that modifies their sensitivity to intracellular Ca2+. In contrast, in human osteosarcoma cells, changes in extracellular pH as well as mitochondrial uncoupling did not exert any inhibitory effects on CCE.     

Green fluorescent protein as a noninvasive intracellular pH indicator.

It was found that the absorbance and fluorescence of green fluorescent protein (GFP) mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells. pH titrations of purified recombinant GFP mutants indicated >10-fold reversible changes in absorbance and fluorescence with pKa values of 6.0 (GFP-F64L/S65T), 5.9 (S65T), 6.1 (Y66H), and 4.8 (T203I) with apparent Hill coefficients of 0.7 for Y66H and approximately 1 for the other proteins. For GFP-S65T in aqueous solution in the pH range 5-8, the fluorescence spectral shape, lifetime (2.8 ns), and circular dichroic spectra were pH independent, and fluorescence responded reversibly to a pH change in <1 ms. At lower pH, the fluorescence response was slowed and not completely reversed. These findings suggest that GFP pH sensitivity involves simple protonation events at a pH of >5, but both protonation and conformational changes at lower pH. To evaluate GFP as an intracellular pH indicator, CHO and LLC-PK1 cells were transfected with cDNAs that targeted GFP-F64L/S65T to cytoplasm, mitochondria, Golgi, and endoplasmic reticulum. Calibration procedures were developed to determine the pH dependence of intracellular GFP fluorescence utilizing ionophore combinations (nigericin and CCCP) or digitonin. The pH sensitivity of GFP-F64L/S65T in cytoplasm and organelles was similar to that of purified GFP-F64L/S65T in saline. NH4Cl pulse experiments indicated that intracellular GFP fluorescence responds very rapidly to a pH change. Applications of intracellular GFP were demonstrated, including cytoplasmic and organellar pH measurement, pH regulation, and response of mitochondrial pH to protonophores. The results establish the application of GFP as a targetable, noninvasive indicator of intracellular pH.     

Activation by acidic pH of CLC-7 expressed in oocytes from Xenopus laevis

ClC chloride channels are important in diverse physiological functions such as transepithelial transport, cell volume regulation, excitability, and acidification of intracellular organelles. We have investigated the expression of CLC-7 in oocytes from Xenopus laevis with the two electrode voltage clamp technique and Western blot analysis. Using a specific antibody against CLC-7, we found an approximately 80 kDa protein in oocytes, previously injected with CLC-7-cRNA. In voltage clamp experiments on ClC-7-cRNA-injected oocytes, no current changes were detected at normal pH (7.4). However, acidification of the Ringer solution to pH values between 6 and 4 revealed strong currents which reversed at about -15 mV (30 mV positive to the normal resting potential) and showed strong outward rectification. We therefore suggest that ClC-7 in oocytes is a functional chloride current at acidic pH. Since ClC-7 is also found in neuronal tissues and was upregulated in a rat pain model, we suggest a role of CLC-7 also for nociception and pain.     

pH and calcium regulate the water permeability of aquaporin 0

Aquaporins increase the water permeability in many cell types across many species. We investigated the effects of external pH and Ca(2+) on water permeability of Xenopus oocytes injected with aquaporin cRNA by measuring the rate of swelling in hypotonic solutions. Lowering pH to 6.5 increased the water permeability of aquaporin (AQP0) 3.4 +/- 0.4-fold. Diethylpyrocarbonate pretreatment increased water permeability 4.2 +/- 0.5-fold and abolished pH sensitivity, suggesting that the pH regulation is mediated by an external histidine. Lowering Ca(2+) increased water permeability 4.1 +/- 0. 4-fold. The effects of Ca(2+) and pH each required the presence of histidine 40, indicating a critical role of this amino acid in facilitating the modulation of water permeability. Clamping intracellular Ca(2+) at high or low values abolished sensitivity to external Ca(2+), suggesting that Ca(2+) acts at an internal site. Three different calmodulin inhibitors each increased AQP0 water permeability, suggesting that Ca(2+) may act through calmodulin. None of the above altered the water permeability induced by AQP1 or AQP4. Because the greatest change in AQP0 water permeability is in the normal pH range found in the lens (7.2-6.5), this paper provides evidence for regulation of an aquaporin by pH under physiological conditions.     

Calcium mediates the light-induced decrease in maintained K+ current in Limulus ventral photoreceptors

In addition to increasing the conductance to sodium, light reduces the maintained voltage-dependent potassium current (iK) in Limulus ventral photoreceptors. We have investigated the mechanism underlying this long-lasting decrease in ik. Intracellular injection of calcium produced a similar reduction of the voltage-dependent outward current. This reduction was not due to an activation of the voltage-dependent inward current (iin) because calcium injection reduced the outward current even under conditions where iin was blocked with Ni2+, and because calcium injection produced a decrease in conductance, as measured from the slope of the instantaneous i-V curve. The effect of light on ik could be blocked by injection of the calcium buffer EGTA (pCa 7.1) to an intracellular concentration of 50-70 mM. Even larger injections of the pH buffer MOPS (100-200 mM) did not reduce the effect of light on ik. These experiments show that intracellular free calcium (Cai2+) can reduce ik. Furthermore, since Cai2+ is known to increase in light, our results are consistent with the hypothesis that calcium is the internal transmitter for the light-induced decrease in ik.