Ciliary membrane vesicles of paramecium contain the voltage-sensitive calcium channel.

Abstract

Calcium influx into ciliary membrane vesicles from paramecia was measured by using arsenazo MI as metallochromic indicator and as intravesicular calcium trap. Influx was rapid and reversible. In ciliary membrane vesicles from paramecium pawn mutants which lack the voltage-sensitive calcium channel, calcium influx was significantly less than in wild-type paramecia. These data demonstrate that the voltage-dependent calcium channel of paramecia is functional in a cell-free system and available for biochemical studies.In response to various environmental stimuli, the paramecium reacts by altering its swimming behavior. The well-known avoiding reaction of paramecia consists of a short period of reversal ofthe ciliary beat, which causes the cell to back away from mechanical, chemical, or other stimulations. This behavioral pattern is correlated with an increase in the internal Ca2" concentration, resulting from the calcium influx associated with a Ca/K action potential (1). The calcium inward current is carried through voltage-sensitive ion channels occurring virtually exclusively in the surface membrane of the cilia (2, 3). Voltagedependent calcium currents with effects on many cellular functions are widespread in metazoan cells, such as in muscle, in nerve cell bodies, or at synapses (4). In fact, in nerve cell bodies and in synapses, which are concerned with information processing, calcium currents seem to play a far more important role than is apparent from studies on axons alone (4).Much of what is known about voltage-dependent calcium currents has come from extensive electrophysiological studies ofHelix (5), Aplysia (6), and Paramecium (7). Because inhibitors that specifically block voltage-dependent calcium channels in Paramecium are not available, a new approach was taken by Kung and associates (8), who isolated mutants of Paramecium with defined defects in excitability and behavior-for example, pawn mutants that apparently lack the calcium channel and are unable to reverse the ciliary beat. This has permitted genetic dissection of the components of the Ca/K action potential (9).Recently, biochemical studies on paramecia have become feasible because techniques for mass culturing wild types and mutants in axenic medium have been developed (10). The exclusive location of the calcium channel in the cilia facilitates production of a membrane fraction greatly enriched in calcium channels. By using the metallochromic dye arsenazo III (AIII) as a calcium indicator as well as an intravesicular calcium trap, we have found evidence that vesicles of the excitable ciliary membrane of paramecia are permeable to Ca2'. More significantly, using identical vesicles from pawn mutants as control, we found compelling evidence that this calcium permeability is a direct result ofthe voltage-sensitive calcium channels ofthe ciliary membrane. MATERIALS AND METHODSPreparation of Ciliary Membrane Vesicles. Paramecium tetraurelia 51 s (wild type) and the pawn mutants d4-94 (a single mutant, genotype pwA/PwA) and pawn A/pawn B (...

<i>Paramecium caudatum</i> Avoids from Naloxone

“…Regarding that Magnesium is a natural calcium antagonist and a potent L-type calcium channel inhibitor [28] rationally the usage of MgSO 4 antagonized the Ca influx signal in the organisms. In addition, considering that the depolarizing Ca 2+ inward current is coupled to the generation of cGMP [24,25,26], the works agree with ours that blockade of Ca channels or cGMP pathway significantly reverse the L-arginine plus naloxone response. Although the role of cGMP has not been elucidated in most systems, it mediates many NO physiological effects as a signaling molecule in the nervous and cardiovascular systems [11,29].…”

“…Our previous results have also implicated NO in the morphine effect by NADPHdiaphorase cytochemistry [1]. It has been postulated that several NO-sensitive targets, including guanylyl cyclase, potassium channels and voltage gated calcium channels [21,22,23,24,25] are stimulated in invertebrates. Other studies using the ciliate protozoan Stentor have demonstrated that these types of microorganisms express a G protein-mediated response to morphine when stimulated mechanically [26].…”

One-way calcium spill-over during signal transduction in Paramecium cells: from the cell cortex into cilia, but not in the reverse direction

“…All evidences favour Ca 2+ as the relevant signal, whereas formation of cGMP during ciliary reversal in Paramecium [50,54,55] may exert a modulatory effect only [45,[56][57][58][59][60]. In Paramecium, guanylate cyclase is distributed over the ciliary membrane [61], but cGMP rises during ciliary reversal induction only with considerable delay [56,62], at least ∼10 times slower than [Ca 2+ ] [55].…”

Immunolocalization of protein phosphatase type 1 in Paramecium cells using antibodies against recombinant protein and peptides.

“…Data obtained thus far indicate a possible role of phosphorylation/dephosphorylation cycles (Schultz et al, 1990). Ciliary reversal is triggered by increased free Ca2+ concentrations via activation of voltage-dependent Ca2+ channels localized in ciliary membranes (Cohen et al, 1990;Schultz et al, 1990;Thiele and Schultz, 1981;Eckert and Brehm, 1979).…”