Changes in functioning of rat submandibular salivary gland under streptozotocin-induced diabetes are associated with alterations of Ca2+ signaling and Ca2+ transporting pumps.

Xerostomia and pathological thirst are troublesome complications of diabetes mellitus associated with impaired functioning of salivary glands; however, their cellular mechanisms are not yet determined. Isolated acinar cells were loaded with Ca2+ indicators fura-2/AM for measuring cytosolic Ca2+ concentration ([Ca2+]i) or mag-fura-2/AM-inside the endoplasmic reticulum (ER). We found a dramatic decrease in pilocarpine-stimulated saliva flow, protein content and amylase activity in rats after 6 weeks of diabetes vs. healthy animals. This was accompanied with rise in resting [Ca2+]i and increased potency of acetylcholine (ACh) and carbachol (CCh) but not norepinephrine (NE) to induce [Ca2+]i transients in acinar cells from diabetic animals. However, [Ca2+]i transients mediated by Ca2+ release from ER stores (induced by application of either ACh, CCh, NE, or ionomycin in Ca2+-free extracellular medium) were decreased under diabetes. Application of inositol-1,4,5-trisphosphate led to smaller Ca2+ release from ER under the diabetes. Both plasmalemma and ER Ca2+-ATPases activity was reduced and the latter showed the increased affinity to ATP under the diabetes. We conclude that the diabetes caused impairment of salivary cells functions that, on the cellular level, associates with Ca2+ overload, increased Ca2+-mobilizing ability of muscarinic but not adrenergic receptors, decreased Ca2+-ATPases activity and ER Ca2+ content.

Influence of external pH on two types of low-voltage-activated calcium currents in primary sensory neurons of rats.

The influence of extracellular pH (pH(o)) on low-voltage-activated calcium channels of acutely isolated DRG neurons of rats was examined using the whole cell patch-clamp technique. It has been found that in the neurons of middle size with capacitance C=60+/-4.8 pF (mean+/-S.E., n=8) extracellular acidification from pH(o) 7.35 to pH(o) 6.0 significantly and reversibly decreased LVA calcium current densities by 75+/-3.7%, shifted potential for half-maximal activation to more positive voltages by 18.7+/-0.6 mV with significant reduction of its voltage dependence. The half-maximal potential of steady-state inactivation shifted to more positive voltages by 12.1+/-1.7 mV (n=8) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of middle size have midpoint pK(a)=6.6+/-0.02 and hill coefficient h=0.94+/-0.04 (n=5). In small cells with capacitance C=26+/-3.6 pF (n=5), acidosis decreased LVA calcium current densities only by 15.3+/-1.3% and shifted potential for half-maximal activation by 5.5+/-1.0 mV with reduction of its voltage dependence. Half-maximal potential of steady-state inactivation shifted to more positive voltages by 10+/-1.6 mV (n=4) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of small size have midpoint pK(a)=7.9+/-0.04 and hill coefficient h=0.25+/-0.1 (n=4). These two identified types of LVA currents besides different pH sensitivity demonstrated different kinetic properties. The deactivation of LVA currents with weak pH sensitivity after switching off depolarization to -30 mV had substantially longer decay time than do currents with strong pH sensitivity (tau(d) approximately 5 ms vs. 2 ms respectively). It was found that the prolongation of depolarization steps slows the subsequent deactivation of T-type currents in small DRG neurons. Deactivation traces in these neurons were better described by the sum of two exponentials. Thus, we suppose that T-type channels in small DRG neurons are presented mostly by alpha1I subunit. We suggest that these two types of LVA calcium channels with different sensitivity to external pH can be differently involved in the origin of neuropathic changes.

Calcium clamp in single nerve cells.

Free calcium concentration in isolated single neurons was clamped using a new technical approach based on a feed-back connection between the Fura-2 fluorescence signal measuring the intracellular Ca2+ concentration ([Ca2+]i) and iontophoretic current injecting Ca2+ into the cell. Beginning of [Ca2+]i clamping at a level above the basal one triggered fast (few seconds) current transients equal to injection of 36 +/- 20 microM Ca2+ (for a 0.1 microM change of [Ca2+]i), representing the filling of a fast cytosolic buffer. Continuation of clamping required very small clamping currents (corresponding to injection of 0.39 +/- 0.20 microM.s-1 Ca2+). This value increased proportionally to the magnitude of the change of [Ca2+]i above basal level, indicating the activation of calcium-dependent mechanisms for Ca2+ removal from the cytosol. The described approach allowed measurement, under physiological conditions, of the capacitative and kinetic properties of different Ca-regulating systems functioning in a single nerve cell as well as other types of cells.

Diversity of calcium ion channels in cellular membranes.

Successful introduction of techniques for separation of different ionic currents and recording of single channel activity has demonstrated the diversity of membrane structures responsible for generation of calcium signal during various forms of cellular activity. In excitable cells the electrically-operated calcium channels have been separated into two types functioning in different membrane potential ranges (low- and high-threshold ones). The low-threshold channels are ontogenetically primary and may play a role in regulation of cell development and differentiation. A similar function may also be characteristic of chemically-operated channels in some highly specialized cells (lymphocytes). The high-threshold channels in excitable cells generate an intracellular signal coupling membrane excitation and intracellular metabolic processes responsible for specific cellular reactions (among them retention of traces of previous activity in neurons--"learning"--being especially important). Chemically-operated N-methyl-D-aspartate-channels also participate in this function. The calcium signal can be potentiated by activation of calcium-operated channels in the membranes of intracellular structures, resulting in the liberation of calcium ions from the intracellular stores. Although different types of calcium channels have some common features in their structure which may indicate their genetic similarity, their specific properties make them well suited for participation in a wide range of cellular mechanisms.

Low-voltage activated calcium channels: achievements and problems.

Low-voltage activated Ca2+ channels, which possess unique properties quite different from those of common (high-voltage activated) channels, were discovered 15 years ago but the first alpha1 subunit has only recently been identified which might provide their structural basis. However, simultaneously, extensive data are being accumulated on the functional diversity of low-voltage activated Ca2+ currents with regard to their pharmacological sensitivity, ionic selectivity, activation and inactivation kinetics. Such diversity corresponds to equally prominent heterogeneity in the location and function of the channels. This commentary summarizes the data available in an attempt to predict a possibly wider structural subdivision of low-voltage activated Ca2+ channels into subtypes.

Spatial heterogeneity of caffeine- and inositol 1,4,5-trisphosphate-induced Ca2+ transients in isolated snail neurons.

Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ release was examined in neurons isolated from the mollusc Helix pomatia using Ca2+ indicator fura-2 and fluorescent digital-imaging microscopy technique. Extracellular application of caffeine caused a fast and pronounced augmentation of [Ca2+]i whose amplitude and kinetics differ in the centre of the cell and near its membrane. Mean values of caffeine-induced increase of [Ca2+]i were 0.97 +/- 0.11 microM at the periphery and 0.53 +/- 0.13 microM in the centre. The rates of rise and relaxation of caffeine-evoked [Ca2+]i transients were faster near the membrane. Pressure injection of inositol, 1,4,5-trisphosphate into the same neurons produced an abrupt and significant increase of [Ca2+]i in the centre (mean value of inositol 1,4,5-trisphosphate-induced elevation = 0.55 +/- 0.11 microM) while the response was smaller or even absent near the cellular membrane. Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ transients did not affect each other. The data obtained indicate that in snail neurons these two calcium pools are not overlapping and at least some part of the caffeine-sensitive store is located close to the cellular membrane and that the inositol 1,4,5-trisphosphate-sensitive one is located in the centre of the cell.

Potassium outward current dependent on extracellular calcium in snail neuronal membrane.

In experiments on isolated intracellularly dialysed neurons of the snail Helix pomatia a component of delayed inactivating potassium outward current depending on the presence of Ca2+ ions in the extracellular medium has been distinguished, which differs from the already known potassium current sensitive to intracellular calcium ions. This component decreases with a decrease in extracellular calcium (in the range of 10(-2) - 10(-5) M); it is not affected or even increased by intracellular introduction of ethyleneglycolbis(aminoethylether)tetra-acetate (10 mM) or fluoride ions (77 mM) and can be blocked by addition of 1.5 mM cobalt ions to the extracellular solution. Contrary to the slow rising potassium current dependent on intracellular calcium, this current has a fast rising phase (several milliseconds) and time-dependent inactivation. The inactivation depends on extracellular potassium ions: it slowed down when [K+]out is increased in the range of 1-10 mM. Extracellular application of calmodulin blockers calmidazolium (6.5 X 10(-7) M) and chlorpromazine (2.5 X 10(-6) M) selectively inhibits the potassium current dependent on intracellular calcium but does not affect that dependent on external calcium. Tetraethylammonium (10 mM) depresses the latter current on both intra- and extracellular application, the former being more effective. The existence of a special type of potassium channel sensitive to extracellular calcium ions is postulated.

Polarization of primary afferent terminals of lumbosacral cord elicited by the activity of spinal locomotor generator.

The changes of electrical polarization of primary afferent terminals in the lumbosacral spinal cord have been investigated on immobilized, decorticated and spinal cats during fictive locomotion. Fictive locomotion was spontaneous or provoked by stimulation of either dorsal root or dorsal funiculi in the lumbar segments. The activation of the locomotor generator and appearance of fictive locomotion were always associated with a sustained dorsal root hyperpolarization. On the background of this positivity periodic negative dorsal root potential oscillations appeared synchronously with efferent discharges in motor hind-limb nerves. These periodic waves of primary afferent depolarization occurred in phase in different ipsilateral lumbosacral segments. On the contralateral side the periodic changes in dorsal root potential were out of phase during fictive stepping and in phase during fictive galloping. The use of Wall's technique has shown that tonic and periodic changes in dorsal root potential reflect the changes occurring in polarization of central terminals of cutaneous and muscle (Ia and Ib) groups of afferent fibres. It is concluded that the level of electrical polarization of primary afferent terminals is determined directly by the activity of the spinal locomotor generator; activation of the generator is followed by hyperpolarization of primary afferent terminals. By so modulating the polarization of afferent terminals, the locomotor generator can perform tonic and phase-dependent selection of afferent information.

Glucocorticoid modulation of calcium currents in growth hormone 3 cells.

Clonal malignant pituitary growth hormone 3 cells were used for the analysis of the influence of hydrocortisone and dexamethasone on voltage-gated calcium currents and hormone secretion. The whole-cell patch-clamp technique was used. The presence of low-threshold inactivating and high-threshold persisting components in the total calcium current was shown; they could be separated at less negative holding potential level. Some increase in current densities of both components was observed as early as 30 min after treatment with 10(-6) mol/l glucocorticoids. The increase was maximal for both types of currents after 2 h of incubation; however, the high-threshold component was affected much more strongly (current density increased by more than four-fold) than the low-threshold one (current density increased by about a three-fold). Potentiation of currents was blocked by actinomycine D (10(-4) M), suggesting that protein synthesis was required. A substantial increase in growth hormone secretion (measured by radioimmunoassay method) was observed in the same cells after 2 h of incubation with hydrocortisone, while the secretion of prolactin remained even slightly depressed.

Modulation of calcium current by calmodulin antagonists.

The short-term effects of bath applied calmodulin antagonists--chlorpromazine, trifluoperazine and calmidazolium (R24571)--on potential-dependent calcium channels in the membrane of intracellularly perfused snail neurons were studied in voltage clamp conditions. All the drugs affected the calcium inward current peak value, the effects being reversible and dependent on the concentration used. Submicromolar concentrations (0.1-1 microM) increased the current amplitude (the maximal effect was on the average 20% at 0.5 microM), whereas higher concentrations inhibited the current. Analysis of the dose-effect curve for the blockade suggests positive cooperativity in the interaction of the drugs with the channel; experimental data on chlorpromazine action (10-100 microM) are well approximated by a binding curve for two molecules with the effective Kd = 70 microM. The efficiency of the blockade depended neither on the current-carrying cations (calcium or barium) nor on the intracellular introduction of 10 mM EGTA. The presence of calmodulin antagonists influenced the blockade of the calcium current by inorganic blockers: 50 microM chlorpromazine decreased the Kd value from 90 to 50 microM for the current blockade by Cd ions. It is suggested that calmodulin antagonists interact with two sites in the calcium channel, with high and low binding affinity (responsible for enhancement and inhibition of the current, respectively). The interaction induces changes in binding of penetrating cations in the channel, thereby producing modulation of the calcium current amplitude.

Long-term depolarization changes morphological parameters of PC12 cells.

It is well known that neuronal differentiation is strongly dependent on the intracellular level of free calcium ions ([Ca2+]i). In the present study the morphological and intracellular free calcium concentration changes were compared on PC12 pheochromocytoma cells cultured in control conditions and in a medium with high KCl level. Culturing PC12 cells in a medium with 20-30 mM KCl deprived of nerve growth factor supported cell proliferation and rapid growth of small neurite-like processes. However, their lengths did not increase with prolongation of the time of culturing. During culturing with 40 mM KCl the growth of these processes became blocked; the cells stopped proliferating and showed signs of degeneration. Measurements of [Ca2+]i level during the first days of PC12 cells culturing in a hyperpotassium medium indicate that such changes in this level could be an important factor in the induction of the observed morphological alterations; however, other effects induced by membrane depolarization may also be responsible for them.

Comparative analysis of ionic currents in the somatic membrane of embryonic and newborn rat sensory neurons.

Inward currents in the somatic membrane of dissociated rat dorsal root ganglion neurons have been studied in two groups of animals (17 days of embryonic development and the first day after birth) by suction pipette (whole-cell configuration) and voltage-clamp techniques. Altogether 157 neurons were examined. Four components in the inward currents have been identified: fast tetrodotoxin-sensitive (INaf) and slow tetrodotoxin insensitive (INas) sodium, low-(ICal) and high-threshold (ICah) calcium currents. The percentage of neurons demonstrating four types of inward currents INaf, INas, ICal, ICah increased from 21% in embryo to 61% in newborn. The percentage of neurons with INaf, INas and ICah increased from 4% in embryonic to 14% in the first day after birth. The percentage of cells with INaf, ICal and ICah (without tetrodotoxin-insensitive INas) decreased from 56 to 11% in embryo and newborn rats, respectively. A statistically significant linear correlation was found between the densities of INaf and ICah currents for both ages. A correlation also occurred between the densities of INas and ICah. A reciprocal relation between the densities of both types of calcium currents and the size of cell soma was found in the neurons with all four types of inward currents from newborn animals. A comparison of these data with previous study of inward currents during postnatal development indicates that the most dramatic changes in their distributions and mean densities takes place some time after the birth of the animals.

Ionic mechanisms of electrical excitability in rat sensory neurons during postnatal ontogenesis.

Inward currents in the somatic membrane of rat dorsal root ganglion neurons have been studied in three groups of animals (8-10, 45-50 and 90-100 days of postnatal development) by intracellular perfusion and voltage clamp techniques. Altogether, 228 neurons have been examined (76 in each age group). Four components have been identified in the inward current: fast tetrodotoxin-sensitive (IfNa) and slow tetrodotoxin-insensitive (IsNa) sodium, low threshold (IlCa) and high threshold (IhCa) calcium currents. The existence of certain types of inward currents in the somatic membrane allowed the heterogeneous population of all the investigated neurons to be divided into six homogeneous subpopulations. A significant decrease in the percentage of neurons was found, demonstrating four types of inward currents (IfNa, IsNa, IlCa, IhCa) during postnatal ontogenesis. The percentage of cells in the subpopulation showing only IfNa and IhCa increased from 26% in the first age group to 43% in the second and 62% in the third age groups. Correlations between densities of various types of inward currents were studied. A linear relationship was found between the densities of IhCa and IsNa in subpopulations of neurons with IsNa. An inverse dependence was observed between the densities of IfNa and IhCa in cells showing only two current components. In all cells studied a "washout" of IhCa was observed during intracellular dialysis with saline solutions. Recovery of calcium conductance produced by intracellular application of an cAMP-ATP-Mg2+ complex was different in neurons with various inward current combinations. Intracellular introduction of cAMP-ATP-Mg2+ failed to restore IhCa in most cells of the second and third age groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Different action of ethosuximide on low- and high-threshold calcium currents in rat sensory neurons.

The effects of ethosuximide on calcium channels were studied on dorsal root ganglion neurons from one-day-old rats using the patch-clamp technique. Bath application of ethosuximide induced dose-dependent and reversible suppression of calcium currents without affecting their time-course. Substantial differences between the effects of ethosuximide on the low-threshold and high-threshold (T- and L-) currents were observed. Ethosuximide reduced the T-current with greater potency than the L-current (Kd for T-current is 7 microM vs 15 microM for L-current). This relative specificity of its action still remained if applied at concentrations up to 1 mM. These data support the hypothesis according to which the anti-epileptic action of ethosuximide is related to reduction of the low-threshold calcium currents in sensory neurons.

Effect of streptozotocin-induced diabetes on the activity of calcium channels in rat dorsal horn neurons.

We have previously found that spinal dorsal horn neurons from streptozotocin-diabetic rats, an animal model for diabetes mellitus, show the prominent changes in the mechanisms responsible for [Ca2+]i regulation. The present study aimed to further characterize the effects of streptozotocin-induced diabetes on neuronal calcium homeostasis. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2AM-loaded dorsal horn neurons from acutely isolated spinal cord slices using fluorescence technique. We studied Ca2+ entry through plasmalemmal Ca2+ channels during potassium (50 mM KCl)-induced depolarization. The K+-induced [Ca2+]i elevation was inhibited to a different extent by nickel ions, nifedipine and omega-conotoxin suggesting the co-expression of different subtypes of plasmalemmal voltage-gated Ca2+ channels. The suppression of [Ca2+]i transients by Ni2+ (50 microM) was the same in control and diabetic neurons. On the other hand, inhibition of [Ca2+]i transients by nifedipine (50 microM) and omega-conotoxin (1 microM) was much greater in diabetic neurons compared with normal animals. These data suggest that under diabetic conditions the activity of N- and L- but not T-type voltage-gated Ca2+ channels substantially increased in dorsal horn neurons.

Nifedipine-induced morphological differentiation of rat pheochromocytoma cells.

Extension of neuronal processes is fundamental for the establishment of the intricate network of the nervous system. The rat PC12 pheochromocytoma cell line is a well-known model system for the study of neuronal differentiation. In the present study the effect of a high-voltage-activated calcium channel blocker, nifedipine, on the cell differentiation was investigated. In cells cultured in a modified medium containing nifedipine (at 2.5 microM or 5.0 microM) a decrease of mitotic activity took place on the third day of culturing. An increase of total length and number of processes occurred on the fifth day. A higher concentration of nifedipine (10.0 microM) considerably suppressed vital functions of the cells. Retraction of processes together with blocking of proliferation activity was observed. In parallel with these changes, a reduction of [Ca2+]i from 100 +/- 6 nM to 50 +/- 2 nM was detected on the second day of cultivation in 2.5 microM and 5.0 microM nifedipine containing medium. At 10.0 microM concentration of nifedipine in the medium a decrease in [Ca2+]i was observed on the first day only (to 76 +/- 8 nM); then the level of free calcium concentration rose dramatically. The data obtained allow us to suggest that the decrease of [Ca2+]i during first two days of PC12 cells culturing in a nifedipine-containing medium could be a factor which stimulated their morphological differentiation.

Intracellular protein kinase and calcium inward currents in perfused neurones of the snail Helix pomatia.

Changes in the amplitude of the calcium inward current caused by intracellular administration of tolbutamide (an inhibitor of the cyclic AMP-dependent protein kinase activity) or catalytic subunits of cAMP-dependent protein kinases from rabbit myocardium were studied on internally perfused nerve cells of the snail, Helix pomatia. Intracellular administration of 7 mM tolbutamide caused a rapid decline of the amplitude of the calcium current that had been stabilized by theophylline; the effect was practically completely reversible. In contrast, addition to the perfusing solution of exogenous catalytic subunits of cyclic AMP-dependent protein kinases (about 0.7 microM of protein) together with 2 mM adenosine 5'-triphosphate and 3 mM MgCl2, led to stabilization of the calcium conductance of the cell membrane or restored it if it had declined during the perfusion with basic solution. The effect depended largely on the presence of adenosine 5'-triphosphate. Its time course was very slow (dozens of minutes) due probably to slow diffusion of the protein inside the cell. Heat-inactivated catalytic subunits did not produce such a stabilizing or restoring action on the calcium conductance. The results substantiate the suggestion that the normal functioning of calcium channels depends on phosphorylation catalyzed by cyclic AMP-dependent protein kinases.

Changes in calcium signalling in dorsal horn neurons in rats with streptozotocin-induced diabetes.

Intracellular calcium signalling was studied in the dorsal horn from neurons of rats with streptozotocin-induced diabetes versus control animals. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2 acetoxymethyl ester-loaded dorsal horn neurons from acutely isolated spinal cord slices using a fluorescence technique. The recovery of depolarization-induced [Ca2+]i increase was delayed in diabetic neurons compared with normal animals. In normal neurons, [Ca2+]i after the end of KCl depolarization recovered to the basal level monoexponentially with a time constant of 8.0+/-0.5 s (n = 23), while diabetic neurons showed two exponentials in the [Ca2+]i recovery. The time constants of these exponentials were 7.2+/-0.5 and 23.0+/-0.6 s (n = 19), respectively. The amplitude of calcium release from caffeine-sensitive endoplasmic reticulum calcium stores became significantly smaller in diabetic neurons. The amplitudes of [Ca2+]i transients evoked by 30 mM caffeine were 268+/-29 nM (n = 13) and 31+/-9 nM (n = 17) in control and diabetic neurons, respectively. We conclude that streptozotocin-induced diabetes is associated with prominent changes in the mechanisms responsible for [Ca2+]i regulation, which presumably include a slowdown of Ca2+ elimination from the cytoplasm by the endoplasmic reticulum.

Characterization of hypothalamic low-voltage-activated Ca channels based on their functional expression in Xenopus oocytes.

Ca-channel currents expressed in Xenopus oocytes by means of messenger RNA extracted from rat thalamohypothalamic complex were studied using the double microelectrode technique. Currents were recorded in Cl(-)-free extracellular solutions with 40 mM Ba2+ as a charge carrier. In response to depolarizations from a very negative holding potential (Vh = -120 mV), inward Ba2+ current activated at around -80 mV, peaked at -30 to -20 mV and reversed at +50 mV indicating that it may be transferred through the low voltage-activated calcium channels. The time-dependent inactivation of the current during prolonged depolarization to -20 mV was quite slow and followed a single exponential decay with a time-constant of 1550 ms and a maintained component constituting 30% of the maximal amplitude. The current could not be completely inactivated at any holding potential. As expected for low voltage-activated current, steady-state inactivation curve shifted towards negative potentials. It could be described by the Boltzmann equation with half inactivation potential -78 mV, slope factor 15 mV and maintained level 0.3. Expressed Ba2+ current could be blocked by flunarizine with Kd = 0.42 microM, nifedipine, Kd = 10 microM, and amiloride at 500 microM concentration. Among inorganic Ca-channel blockers the most potent was La3+ (Kd = 0.48 microM) while Cd2+ and Ni2+ were not very discriminative and almost 1000-fold less effective than La3+ (Kd = 0.52 mM and Kd = 0.62 mM, respectively). Our data show that messenger RNA purified from thalamohypothalamic complex induces expression in the oocytes of almost exclusively low voltage-activated calcium channels with voltage-dependent and pharmacological properties very similar to those observed for T-type calcium current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.

Divalent cation selectivity of the subtypes of low voltage-activated Ca2+ channels in thalamic neurons.

LVA Ca2+ current in isolated associative neurons from the laterodorsal thalamic nucleus of 14- to 17-day-old rats was dissected into two, 'fast' and 'slow', components based on the difference in the kinetics of inactivation. The selectivity of the channel responsible for the fast LVA current for Ca2+, Sr2+ and Ba2+ (I(Ca):I(Sr):I(Ba) = 1.0:1.23:0.94) as well as the shifts of the I-V produced by these ions were found to be almost identical to those observed for LVA channels in other preparations. The channel responsible for the slow LVA current showed selectivity more characteristic of HVA Ca2+ channels (I(Ca):I(Sr):I(Ba) = 1.0:2.5:3.4), although the ability of Ca2+, Sr2+ and Ba2+ to shift its voltage dependence remained the same as for the fast channel.