Prolactin storage in a clonal strain of rat pituitary tumor cells is cell-cycle dependent

GH4C1 cells (CH cells) are a clonal strain of rat pituitary tumor cells which secrete prolactin. We measured intracellular prolactin at different stages of the cell cycle using flow microfluorometry. Prolactin was stained by an indirect immunocytochemical technique using fluorescein isothiocyanate (FITC)-conjugated antiserum, and DNA was stained simultaneously with propidium iodide. We found that prolactin storage in GH cells was cell-cycle dependent; prolactin storage increased as cells passed from G1 to S to G2 + M. We have shown previously that insulin and 17 beta-estradiol act synergistically to increase intracellular prolactin three- to sevenfold and slow the rate of cell growth to approximately 70% of control cells. In this study we observed that insulin and estradiol increased prolactin storage at each stage of the cell cycle but did not affect the cell-cycle distribution of the population even though cell growth was slowed. We conclude that insulin and estradiol did not increase prolactin storage by affecting the cell-cycle distribution of the population.     

Sodium channel inactivation from closed states: evidence for an intrinsic voltage dependency.

The time course of Na channel inactivation from closed states was determined on inside-out excised patches from neuroblastoma N1E 115. Closed-state inactivation develops as a single exponential with mean time constants of 66.4 ms at -80 mV, 29.6 ms at -70 mV, 20.1 ms at -60 mV, and 15.1 ms at -50 mV. Corresponding mean steady-state values of the fitted exponentials were 0.321, 0.098, 0.035, and 0. Closed-state inactivation, in general, should develop either with a delay or as more than one exponential, depending on which closed state(s) directly inactivate. The absence of additional components cannot be attributed to a rate of exchange between closed states too rapid to detect. The time course is simply accounted for if all closed states directly inactivate and do so with the same rate constant for each closed state, suggesting that those conformational changes constituting the transitions between closed states have little effect on the structural components involved in inactivation. Closed to inactivated rate constants ranged from a mean of 0.0108 ms-1 at -80 mV to 0.0690 ms-1 at -50 mV. This voltage dependency is entirely intrinsic to closed-state inactivation with closed to inactivated rate constants similar for all closed states. Over the potential range studied nearly all the inactivation is from closed states.     

The quantal gating charge of sodium channel inactivation

Using a very low noise voltage clamp technique it has been possible to record from the squid giant axon a slow component of gating current (I _g ) during the inactivation phase of the macroscopic sodium current (I _Na ) which was hitherto buried in the baseline noise. In order to examine whether this slowI _g contains gating charge that originates from transitions between the open (O) and the inactivated (I) states, which would indicate a true voltage dependence of inactivation, or whether other transitions contribute charge to slowI _g , a new model independent analysis termed isochronic plot analysis has been developed. From a direct correlation ofI _g and the time derivative of the sodium conductance dg _Na/d the condition when only O-I transitions occur is detected. Then the ratio of the two signals is constant and a straight line appears in an isochronic plot ofI _g vs. dg _Na/d . Its slope does not depend on voltage or time and corresponds to the quantal gating charge of the O-I transition (q _h ) divided by the single channel ionic conductance (). This condition was found at voltages above – 10 mV up to + 40 mV and a figure of 1.21e ^– was obtained forq _h at temperatures of 5 and 15°C. At lower voltages additional charge from other transitions, e.g. closed to open, is displaced during macroscopic inactivation. This means that conventional Eyring rate analysis of the inactivation time constant _h is only valid above – 10 mV and here the figure forq _h was confirmed also from this analysis. It is further shown that most of the present controversies surrounding the voltage dependence of inactivation can be clarified. The validity of the isochronic plot analysis has been confirmed using simulated gating and ionic currents.Abbreviations I _g gating current - I _Na sodium ionic current - g _Na macroscopic sodium conductance - single channel conductance - C, O, I closed, open, inactivated state occupancy of channels - g _h quantal charge displaced in a single O-I transition of Na channel - e ^– equivalent electron charge - h index referring to inactivation process - S _l limiting slope in isochronic plot see Eq.(3) - fractional distance, see Fig. 4 and (4, 5) - TMA tetramethylammonium - TTX tetrodotoxin - Tris tris(hydroxymethyl)aminomethane - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid     

Movement of voltage sensor S4 in domain 4 is tightly coupled to sodium channel fast inactivation and gating charge immobilization.

The highly charged transmembrane segments in each of the four homologous domains (S4D1-S4D4) represent the principal voltage sensors for sodium channel gating. Hitherto, the existence of a functional specialization of the four voltage sensors with regard to the control of the different gating modes, i.e., activation, deactivation, and inactivation, is problematic, most likely due to a functional coupling between the different domains. However, recent experimental data indicate that the voltage sensor in domain 4 (S4D4) plays a unique role in sodium channel fast inactivation. The correlation of fast inactivation and the movement of the S4D4 voltage sensor in rat brain IIA sodium channels was examined by site-directed mutagenesis of the central arginine residues to histidine and by analysis of both ionic and gating currents using a high expression system in Xenopus oocytes and an optimized two-electrode voltage clamp. Mutation R1635H shifts the steady state inactivation to more hyperpolarizing potentials and drastically increases the recovery time constant, thereby indicating a stabilized inactivated state. In contrast, R1638H shifts the steady state inactivation to more depolarizing potentials and strongly increases the inactivation time constant, thereby suggesting a preferred open state occupancy. The double mutant R1635/1638H shows intermediate effects on inactivation. In contrast, the activation kinetics are not significantly influenced by any of the mutations. Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H. The time courses of recovery from inactivation and immobilization correlate well in wild-type and mutant channels, suggesting an intimate coupling of these two processes that is maintained in the mutations. These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state. Moreover, the presented data strongly suggest that S4D4 is involved in the control of fast inactivation.     

Sodium channel gating modes during redox reaction.

BACKGROUND/AIMS: Many studies have confirmed that persistent sodium current (I(NaP)) is altered during a redox reaction, but little attention has been paid to transient sodium current (I(NaT)) and its correlation with I(NaP) during the redox reaction. The aim of the study was to investigate the effect of the redox states on the correlation between I(NaT) and I(NaP) in cardiomyocytes. METHODS: I(NaT) and I(NaP) were recorded using whole-cell and cell-attached patch-clamp techniques in guinea pig ventricular myocytes. RESULTS: In whole-cell recordings, dithiothreitol (DTT, 1 mM) simultaneously increased I(NaT) and decreased I(NaP). Hydrogen peroxide (H(2)O(2), 0.3 mM) increased I(NaP) and decreased I(NaT) in a time-dependent manner, which were reversed by DTT (1 mM). In cell-attached recordings, the increasing of I(NaP) and decreasing of I(NaT) induced by H(2)O(2) (0.3 mM) were similarly recovered by DTT (1 mM). H(2)O(2) (0.3 mM) prolonged the action potential (AP) duration of ventricular papillary cells whereas decreased the AP amplitude and maximum rate of depolarization (V(max)) in a time-dependent manner, which were reversed by DTT (1 mM). CONCLUSION: These results indicate that the redox states could modulate the sodium channel gating modes in guinea pig ventricular myocytes.     

Dexamethasone increases potassium channel messenger RNA and activity in clonal pituitary cells.

Glucocorticoid hormones are released as part of the stress response and regulate secretion by the pituitary. Since the activity of ion channels also influences secretion, we examined the effect of the glucocorticoid agonist dexamethasone on ion channel expression. K+ channel mRNA was detected in rat hypothalamus and anterior pituitary, with probes derived from the rat Kv1 gene, a member of the mammalian voltage-gated K+ channel superfamily. High levels were also detected in PRL-secreting clonal (GH3 and GH4C1) rat pituitary cells. Dexamethasone rapidly increased the steady state concentration of Kv1 mRNA in GH3 cells in a dose-dependent manner. This change in gene expression was accompanied by an increase in whole cell voltage-gated K+ current [lk(i)] with similar pharmacology to the Kv1 gene product. Our findings indicate that hormones may act directly on excitable cells to produce long term effects on electrical activity and secretion by regulating K+ channel expression.     

Measurement of calcium channel inactivation is dependent upon the test pulse potential.

We have developed two methods to measure Ca2+ channel inactivation in Lymnaea neurons-one method, based upon the conventional double-pulse protocol, uses currents during a moderately large depolarizing pulse, and the other uses tail currents after a very strong activating pulse. Both methods avoid contamination by proton currents and are unaffected by rundown of Ca2+ current. The magnitude of inactivation measured differs for the two methods; this difference arises because the measurement of inactivation is inherently dependent upon the test pulse voltage used to monitor the Ca2+ channel conductance. We discuss two models that can generate such test pulse dependence of inactivation measurements-a two-channel model and a two-open-state model. The first model accounts for this by assuming the existence of two types of Ca2+ channels, different proportions of which are activated by the different test pulses. The second model assumes only one Ca2+ channel type, with two closed and open states; in this model, the test pulse dependence is due to the differential activation of channels in the two closed states by the test pulses. Test pulse dependence of inactivation measurements of Ca2+ channels may be a general phenomenon that has been overlooked in previous studies.     

Sodium channel gating currents in frog skeletal muscle

Charge movements similar to those attributed to the sodium channel gating mechanism in nerve have been measured in frog skeletal muscle using the vaseline-gap voltage-clamp technique. The time course of gating currents elicited by moderate to strong depolarizations could be well fitted by the sum of two exponentials. The gating charge exhibits immobilization: at a holding potential of -90 mV the proportion of charge that returns after a depolarizing prepulse (OFF charge) decreases with the duration of the prepulse with a time course similar to inactivation of sodium currents measured in the same fiber at the same potential. OFF charge movements elicited by a return to more negative holding potentials of -120 or -150 mV show distinct fast and slow phases. At these holding potentials the total charge moved during both phases of the gating current is equal to the ON charge moved during the preceding prepulse. It is suggested that the slow component of OFF charge movement represents the slower return of charge "immobilized" during the prepulse. A slow mechanism of charge immobilization is also evident: the maximum charge moved for a strong depolarization is approximately doubled by changing the holding potential from -90 to -150 mV. Although they are larger in magnitude for a -150-mV holding potential, the gating currents elicited by steps to a given potential have similar kinetics whether the holding potential is -90 or -150 mV.     

Sodium channel gating currents. Origin of the rising phase

There has been some uncertainty in the past as to the origin of the rising phase of the gating current. We present evidence here that proves that the gating current does not have a rising phase and that the observed rising phase is due to an uncompensated series resistance in the Frankenhaeuser-Hodgkin (F-H) space. When a squid giant axon is bathed in a solution that is 10-20% hyperosmotic with respect to the internal solution, the rising phase of the gating current is eliminated. In parallel with this, a component of the capacity transient (time constant, 20 microseconds) is reduced so that the capacity transient now appears to be closer to a single fast (5-10 microseconds) component. These changes in the capacity transient and gating current occur without altering the amount of charge moved in either. This indicates that the charge is simply redistributed in time. The gating current without a rising phase can still be immobilized by inactivation. Supporting evidence is provided by measuring the accumulation and washout of K+ from the F-H space. It was found that K+ washes out 35% faster when the axon is bathed in hyperosmotic solution. It was estimated that the F-H space thickness (theta) increased 2.5 +/- 0.4-fold (mean +/- SEM) in hyperosmotic solution. Similarly, K+ accumulation in the F-H space was decreased, leading to an estimate of a 5 +/- 1.4-fold increase in theta in hyperosmotic solution. These results are consistent with the simple structural model presented.     

Large scale rearrangement of protein domains is associated with voltage gating of the VDAC channel.

The VDAC channel of the mitochondrial outer membrane is voltage-gated like the larger, more complex voltage-gated channels of the plasma membrane. However, VDAC is a low molecular weight (30 kDa), abundant protein, which is readily purified and reconstituted, making it an ideal system for analyzing the molecular basis for ion selectivity and voltage-gating. We have probed the VDAC channel by subjecting the cloned yeast (S. cerevisiae) VDAC gene to site-directed mutagenesis and introducing the resulting mutant channels into planar bilayers to detect the effects of specific sequence changes on channel properties. This approach has allowed us to formulate and test a model of the open state structure of the VDAC channel. Now we have applied the same approach to analyzing the structure of the channel's low-conducting "closed state" (essentially closed to important metabolites). We have identified protein domains forming the wall of the closed conformation and domains that seem to be removed from the wall of the pore during channel closure. The latter can explain the reduction in pore diameter and volume and the dramatically altered channel selectivity resulting from the channel closure. This process would make a natural coupling between motion of the sensor and channel gating.     

Negatively charged residues in the N-terminal of the AID helix confer slow voltage dependent inactivation gating to CaV1.2

The E462R mutation in the fifth position of the AID (alpha1 subunit interaction domain) region in the I-II linker is known to significantly accelerate voltage-dependent inactivation (VDI) kinetics of the L-type CaV1.2 channel, suggesting that the AID region could participate in a hinged-lid type inactivation mechanism in these channels. The recently solved crystal structures of the AID-CaVbeta regions in L-type CaV1.1 and CaV1.2 channels have shown that in addition to E462, positions occupied by Q458, Q459, E461, K465, L468, D469, and T472 in the rabbit CaV1.2 channel could also potentially contribute to a hinged-lid type mechanism. A mutational analysis of these residues shows that Q458A, Q459A, K465N, L468R, D469A, and T472D did not significantly alter VDI gating. In contrast, mutations of the negatively charged E461, E462, and D463 to neutral or positively charged residues increased VDI gating, suggesting that the cluster of negatively charged residues in the N-terminal end of the AID helix could account for the slower VDI kinetics of CaV1.2. A mutational analysis at position 462 (R, K, A, G, D, N, Q) further confirmed that E462R yielded faster VDI kinetics at +10 mV than any other residue with E462R > E462K approximately E462A > E462N > wild-type approximately E462Q approximately E462G > E462D (from the fastest to the slowest). E462R was also found to increase the VDI gating of the slow CEEE chimera that includes the I-II linker from CaV1.2 into a CaV2.3 background. The fast VDI kinetics of the CaV1.2 E462R and the CEEE + E462R mutants were abolished by the CaVbeta2a subunit and reinstated when using the nonpalmitoylated form of CaVbeta2a C3S + C4S (CaVbeta2a CS), confirming that CaVbeta2a and E462R modulate VDI through a common pathway, albeit in opposite directions. Altogether, these results highlight the unique role of E461, E462, and D463 in the I-II linker in the VDI gating of high-voltage activated CaV1.2 channels.     

K+ channel gating: C-type inactivation is enhanced by calcium or lanthanum outside

Many voltage-gated K⁺ channels exhibit C-type inactivation. This typically slow process has been hypothesized to result from dilation of the outer-most ring of the carbonyls in the selectivity filter, destroying this ring’s ability to bind K⁺ with high affinity. We report here strong enhancement of C-type inactivation upon extracellular addition of 10–40 mM Ca²⁺ or 5–50 µM La³⁺. These multivalent cations mildly increase the rate of C-type inactivation during depolarization and markedly promote inactivation and/or suppress recovery when membrane voltage (V_m) is at resting levels (−80 to −100 mV). At −80 mV with 40 mM Ca²⁺ and 0 mM K⁺ externally, ShBΔN channels with the mutation T449A inactivate almost completely within 2 min or less with no pulsing. This behavior is observed only in those mutants that show C-type inactivation on depolarization and is distinct from the effects of Ca²⁺ and La³⁺ on activation (opening and closing of the V_m-controlled gate), i.e., slower activation of K⁺ channels and a positive shift of the mid-voltage of activation. The Ca²⁺/La³⁺ effects on C-type inactivation are antagonized by extracellular K⁺ in the low millimolar range. This, together with the known ability of Ca²⁺ and La³⁺ to block inward current through K⁺ channels at negative voltage, strongly suggests that Ca²⁺/La³⁺ acts at the outer mouth of the selectivity filter. We propose that at −80 mV, Ca²⁺ or La³⁺ ions compete effectively with K⁺ at the channel’s outer mouth and prevent K⁺ from stabilizing the filter’s outer carbonyl ring.     

A single charged voltage sensor is capable of gating the Shaker K+ channel

We sought to determine the contribution of an individual voltage sensor to Shaker''s function. Concatenated heterotetramers of Shaker zH4 Δ(6–46) wild type (wt) in combination with a neutral S4 segment Shaker mutant (mut) with stoichiometries 2wt/2mut and 1wt/3mut were studied and compared with the 4wt concatenated homotetramer. A single charged voltage sensor is sufficient to open Shaker conductance with reduced delay (<1 ms) and at more hyperpolarized voltages compared with 4wt. In addition, the wt-like slow inactivation of 1wt/3mut was almost completely eliminated by mutations T449V-I470C in its single wt subunit, indicating that the subunits bearing a neutral S4 were unable to trigger slow inactivation. Our results strongly suggest that a neutral S4 segment of Shaker''s subunit is voltage insensitive and its voltage sensor is in the activated position (i.e., ready for pore opening), and provide experimental support to the proposed model of independent voltage sensors with a final, almost voltage-independent concerted step.     

Sodium dependent inositol transport in HL60 cells is not related to Na+/K+ ATPase activity.

In HL60 cells, inositol transport is sodium-dependent but functionally independent of Na+/K+ ATPase activity. This observation has implications for the currently proposed theory for the development of diabetic complications.     

The role of the putative inactivation lid in sodium channel gating current immobilization

We investigated the contribution of the putative inactivation lid in voltage-gated sodium channels to gating charge immobilization (i.e., the slow return of gating charge during repolarization) by studying a lid-modified mutant of the human heart sodium channel (hH1a) that had the phenylalanine at position 1485 in the isoleucine, phenylalanine, and methionine (IFM) region of the domain III-IV linker mutated to a cysteine (ICM-hH1a). Residual fast inactivation of ICM-hH1a in fused tsA201 cells was abolished by intracellular perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The time constants of gating current relaxations in response to step depolarizations and gating charge-voltage relationships were not different between wild-type hH1a and ICM-hH1a(MTSET). The time constant of the development of charge immobilization assayed at -180 mV after depolarization to 0 mV was similar to the time constant of inactivation of I(Na) at 0 mV for hH1a. By 44 ms, 53% of the gating charge during repolarization returned slowly; i.e., became immobilized. In ICM-hH1a(MTSET), immobilization occurred with a similar time course, although only 31% of gating charge upon repolarization (OFF charge) immobilized. After modification of hH1a and ICM-hH1a(MTSET) with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits movement of the domain IV-S4, charge immobilization did not occur for conditioning durations up to 44 ms. OFF charge for both hH1a and ICM-hH1a(MTSET) modified with Anthopleurin-A toxin were similar in time course and in magnitude to the fast component of OFF charge in ICM-hH1a(MTSET) in control. We conclude that movement of domain IV-S4 is the rate-limiting step during repolarization, and it contributes to charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports, these data also suggest that S4 in domain III contributes to charge immobilization only after binding of the inactivation lid.     

Gating current harmonics. I. Sodium channel activation gating in dynamic steady states.

Internally perfused and pronase-treated giant axons were prepared for gating current measurements. Gating current records were obtained under large-amplitude sinusoidal voltage clamp after allowing for settling times into dynamic steady states. The current records were analyzed as functions of the mean membrane potential of the test sinusoid for which the amplitude and frequency were held constant. The nonlinear analysis consisted of determining the harmonic content (amplitudes and phases) of the distorted periodic current records. The most pronounced feature found in the analysis is a dominant second harmonic centered at Emean = +10 mV. A number of other characteristic harmonic behaviors were also observed. The harmonics tend to die away for very small (less than -60 mV) and very large (greater than +72 mV) values of Emean. The harmonic behavior seen in the axonal data is basically different from that seen in gating current simulations generated by the sodium-activation kinetics of standard models, including the Hodgkin-Huxley model. Some of the differences can be reconciled without requiring fundamental changes in the model kinetic schemes. However, the dominant harmonic feature seen in the axonal data cannot be reconciled with the model kinetics without a fundamental change in the models. The axonal data suggest two moving molecular components with independent degrees of freedom whose properties are outlined on the basis of the data presented herein.     

The calcium-binding EF-hand in polycystin-L is not a domain for channel activation and ensuing inactivation

Polycystin-L (PCL) shares high homology with polycystin-2, the product of polycystic kidney disease gene-2. It was previously shown that the PCL forms a non-selective cation channel activated by calcium influx. However, it remains unclear whether calcium activates/inactivates PCL by binding to the EF-hand motif located on the cytoplasmic carboxyl-terminus. Here we obtained two PCL splice variants from liver (PCL-LV, lacking the EF-hand) and testis (PCL-TS, lacking 45 amino acids on the carboxyl tail) using PCR-based approaches. When expressed in Xenopus oocytes and studied using electrophysiology both splice variants exhibited basal cation channel activity and calcium-induced channel activation. While PCL-TS displayed similar activation to PCL, PCL-LV exhibited a three-fold increased activation. All five PCL C-terminal artificial truncation mutants also exhibited basal and calcium-activated channel activities, in particular the mutant T622X lacking the EF-hand was associated with increased activation. Our data demonstrate that the EF-hand and other parts of the carboxyl tail of PCL are not determinants of channel activation/inactivation although the EF-hand seems to be involved in the modulation of these processes.     

The screw-helical voltage gating of ion channels.

In the voltage-gated ion channels of every animal, whether they are selective for K+, Na+ or Ca2+, the voltage sensors are the S4 transmembrane segments carrying four to eight positive charges always separated by two uncharged residues. It is proposed that they move across the membrane in a screw-helical fashion in a series of three or more steps that each transfer a single electronic charge. The unit steps are stabilized by ion pairing between the mobile positive charges and fixed negative charges, of which there are invariably two located near the inner ends of segments S2 and S3 and a third near the outer end of either S2 or S3. Opening of the channel involves three such steps in each domain.     

Single calcium-dependent potassium channels in clonal anterior pituitary cells.

Single Ca2+-dependent K+-channel currents were recorded in intact and excised inside-out membrane patches of the anterior pituitary clone AtT-20/D16-16. The frequency of channel openings and lifetimes depends both on membrane potential and on the Ca2+ concentrations at the inner membrane surface. The curve of the open-state probability of the channel as a function of membrane potential appears to translate along the voltage axis with changes in internal Ca2+ concentration. For Ca2+ concentrations between 10(-7) and 10(-6) M, the shift is consistent with the hypothesis that three Ca2+ ions are required to open a Ca2+-dependent K+ channel. Single channel conductances are estimated to be 124 pS in patches with normal external K+ (5.4 mM) and 208 pS in excised patches with symmetrical K+ (145 mM) across the membrane. Tetraethylammonium (20 mM) added to the cytoplasmic surface reversibly blocks the Ca2+-dependent K+ channel.