Identification of palmitoylation sites within the L-type calcium channel beta2a subunit and effects on channel function.

The hydrophilic beta2a subunit of the L-type calcium channel was recently shown to be a membrane-localized, post-translationally modified protein (Chien, A. J., Zhao, X. L., Shirokov, R. E., Puri, T. S., Chang, C. F., Sun, D. D., Rios, E., and Hosey, M. M. (1995) J. Biol. Chem. 270, 30036-30044). In this study, we demonstrate that the rat beta2a subunit was palmitoylated through a hydroxylamine-sensitive thioester linkage. Palmitoylation required a pair of cysteines in the N terminus, Cys3 and Cys4; mutation of these residues to serines resulted in mutant beta2a subunits that were unable to incorporate palmitic acid. Interestingly, a palmitoylation-deficient beta2a mutant still localized to membrane particulate fractions and was still able to target functional channel complexes to the plasma membrane similar to wild-type beta2a. However, channels formed with a palmitoylation-deficient beta2a subunit exhibited a dramatic decrease in ionic current per channel, indicating that although mutations eliminating palmitoylation did not affect channel targeting by the beta2a subunit, they were important determinants of channel modulation by the beta2a subunit. Three other known beta subunits that were analyzed were not palmitoylated, suggesting that palmitoylation could provide a basis for the regulation of L-type channels through modification of a specific beta isoform.

Roles of a membrane-localized beta subunit in the formation and targeting of functional L-type Ca2+ channels.

We report several unexpected findings that provide novel insights into the properties and interactions of the alpha 1 and beta subunits of dihydropyridine-sensitive L-type channels. First, the beta 2a subunit was expressed as multiple species of 68-72 kDa; the 70-72-kDa species arose from post-translational modification. Second, cell fractionation and immunocytochemical studies indicated that the hydrophilic beta 2a subunit, when expressed alone, was membrane-localized. Third, the beta 2a subunit increased the membrane localization of the alpha 1 subunit and the number of cells expressing L-type Ca2+ currents, without affecting the total amount of the expressed alpha 1C subunit. Expression of maximal currents in alpha 1C/beta 2a cotransfected cells paralleled the time course of expression of the beta subunit. Taken together, these results suggest that the beta subunit plays multiple roles in the formation, stabilization, targeting, and modulation of L-type channels.

[Action of omega-conotoxin on calcium currents of the rat pituitary GH3 cell line]. / Deistvie omega-konotoksina na kal'tsievye toki kletok linii GH3 gipofiza krysy.

Whole-cell modification of the patch clamp method was used to examine the action of omega-CgTX on calcium currents in GH3 pituitary cells. Two quite distinct components of inward calcium currents were observed in the presence of 15 mmol/l of calcium in the external solution. One was activated from the holding potential -80 mV by testing pulses more positive than -50 mV. The shift of the holding potential to -40 mV resulted in the stationary inactivation of this low voltage activated current component. It was found that omega-CgTX activated both low-threshold and high-threshold calcium currents at the first moment of application, but low-threshold current component increased more significantly. Full effect was developed for less than 30 s. Then time decay of currents was comparable with that of the "wash-out" process. Incubation of cells in the growth medium that contained 5 mumol/l omega-CgTX during 2 hour induced an increase in density of both types of calcium currents, then it fell after 2 hours of incubation in the same medium.

Deactivation kinetics of different components of calcium inward current in the membrane of mice sensory neurones.

1. Deactivation kinetics of different components of calcium inward current were analysed by measuring 'tail currents' in intracellularly perfused sensory neurones isolated from dorsal root ganglia of newborn mice. 2. Deactivation of a low-threshold inactivating component had a mono-exponential time course with time constants that decreased as the potential was made more negative, and reached a limiting value of 1.0-1.2 ms at extreme hyperpolarizations. Replacement of Ca2+ by Ba2+ as a charge carrier decreased this value almost twofold. 3. Deactivation of the high-threshold component of calcium current contained two exponential components with time constants of similar potential dependence. The fast time constant became practically constant (0.1-1.2 ms) during repolarizations beyond -50 mV, and the slow time constant became constant (0.80-0.85 ms) at repolarization beyond -70 mV. 4. Replacement of intracellular aspartate with phosphate, or a depolarized holding potential of -40 mV, abolished reversibly the slow component of the tail current; in parallel, the peak current-voltage relation of the current became shifted by 15-20 mV in the depolarizing direction. 5. Extracellular application of the calcium channel agonist Bay K 8644 (2.5-5 mumol/l) did not change deactivation kinetics of the low-threshold current; however, it increased considerably the slow exponential component of high-threshold current deactivation or induced such a component in conditions when the latter was absent before application (Vh = -40 mV; cell perfusion with Tris phosphate). 6. The data obtained are analysed on the basis of the presence of three types of calcium channels in the neuronal membrane which possess similar kinetic mechanisms of activation and deactivation but differ in the activation thresholds and the time constants of transitions between open and closed states. The three calcium channels differ also in interaction with different permeant ions and calcium channel agonists.

[The potential dependence of calcium current deactivation via the somatic membrane of sensory neurons in the mouse]. / Potentsialozavisimost' deaktivatsii kal'tsievykh tokov cherez somaticheskuiu membranu sensornykh neironov myshi.

Potential dependence of calcium inward current deactivation kinetics was studied in the somatic membrane of mouse dorsal root ganglion neurons by intracellular dialysis technique. The decay of the high-threshold calcium current upon repolarization was reasonably described by single-exponential process with the time constant tau less than or equal to 130 microseconds (V = = -80 mV), when the intracellular solution contained tris-PO4, and by two-exponential process (tau congruent to 0.1 and tau = 0.8 divided by ms, V = -80 mV), when the intracellular solution contained Cs-aspartate and EGTA. Both time constants were strongly voltage dependent. The amplitude of the fast component of the tail current had sigmoidal voltage dependence, and the slow component had V-shaped voltage dependence. The low-threshold calcium current deactivation occurs more slowly with high voltage dependent kinetics (tau = 1.1 divided by 1.2 ms, V = -160 mV). A dependence of low-threshold current deactivation time constant on the type of penetrating cation was observed. A kinetic model of calcium current deactivation was proposed considering three types of calcium channels presented in the somatic membrane of the neurons studied.

[Electroregulated sodium channels of the somatic membrane of sympathetic neurons]. / Elektroupravliaemye natrievye kanaly somaticheskoi membrany simpaticheskikh neironov.

Electrically-operated sodium channels in the somatic membrane of isolated neurons from the rat superior cervical ganglion have been studied by means of intracellular dialysis technique under voltage clamp conditions. It was shown that in this preparation sodium currents can be carried by two independent systems of sodium channels. The mathematical analysis of voltage-dependent TTX-sensitive fast sodium currents was performed by the Hodgkin-Huxley formalism; their kinetic properties were compared with those described in other objects. TTX-sensitive sodium channels in the somatic membrane of sympathetic neurons were found to be highly selective for Na+ ions. Kinetic and voltage-dependent characteristics of slow TTX-resistant sodium current were also described. This component of the sodium current was observed only in a few neurons (not more than 2%).

[Deactivation of calcium currents in the soma of spinal ganglion neurons after elimination of the depolarizing shift in the membrane potential]. / Deaktivatsiia kal'tsievykh tokov v some neironov spinal'nykh gangliev pri vykliuchenii depoliarizuiushchego smeshcheniia membrannogo potentsiala.

Kinetic and voltage-dependent characteristics of deactivation of calcium inward currents with the removal of membrane depolarization were studied in the somatic membrane of rat dorsal root ganglion neurons by intracellular dialysis technique. The "tail" of low-threshold calcium current could be described reliably by one exponent with time constant tau 1-1.2-1.8 ms at repolarization to --90 mV. The "tail" of the high-threshold calcium current represented a sum of several exponents; the time constant of the main component tau h was in the range of 250-380 microseconds. tau 1 and tau h remained practically unchanged for repolarization potentials in the subthreshold region; however, they increased if it was in the range of potentials at which the corresponding component of the calcium current started to activate. A dependence of tau 1 and tau h on the duration of depolarizing shift was observed. The results obtained are discussed in the framework of a three-level kinetic model of calcium channels.