a Paramecium Mutant With an Enhanced Sensitivity to Magnesium

Copyright 0 1997 by the Genetics Society of America

Phenotypic and Genetic Analysis of “Chumeleon,” a Paramecium Mutant With an Enhanced Sensitivity to Magnesium

Robin R. Preston and Jocelyn A. Hammond

Department of Physiology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania I9129 Manuscript received November 15, 1996 Accepted for publication April 18, 1997

ABSTRACT Three mutant strains of Paramecium tetraurelia with an enhanced sensitivity to magnesium have been

isolated. These new “Chameleon” mutants result from partial- or codominant mutations at a single locus, Cha. Whereas the wild type responded to 5 mM Mg’+ by swimming backward for 10-15 sec, Cha mutants responded with -30 sec backward swimming. Electrophysiological analysis suggested that this behavior may be caused by slowing in the rate at which a Mg’+-specific ion conductance deactivates following membrane excitation. This would be consistent with an observed increase in the sensitivity of Cha mutants to nickel poisoning, since Ni“ is also able to enter the cell via this pathway. More extensive behavioral analysis showed that Cha cells also overresponded to Na+, but there was no evidence for a defect in intracellular Ca2+ homeostasis that might account for a simultaneous enhancement of both the Mg‘+ and Na+ conductances. The possibility that the Cha locus may encode a specific regulator of the M$+- and Na+-permeabilities is considered.

R ECENT years have witnessed a remarkable reap praisal of the role of the magnesium ion in cells

(GRUBBS and MACUIRE 1987; WHITE and HARTZELL 1989; ROMANI and SCARPA 1992). While magnesium has long been hailed as an essential cofactor for the normal functioning of many hundreds of enzymes, new evidence suggests that intracellular free Mg2+ ( [Mg2’Ii) may also be an important regulator of cell activity.

Most eukaryotic cells maintain [Mg2’Ii at between 0.5 and 0.8 mM against an electrochemical gradient (ALVAREZ-LEEFMANS et al. 1987). This is made possible through the activity of Mgzfspecific transporters, most commonly a Nat/Mg2+ exchanger (FLATMAN 1991; VORMANN and GUNTHER 1993). The reason that cells expend energy to hold [Mg2’Ii at low levels is still de- bated, but one idea is that changes in [Mg”], are used as an intracellular signal. Indeed, LOSTROH and KRAHL

(1974) have proposed that Mg2+ is a second messenger for insulin-receptor binding. Changes in [Mg2’Ii have also been observed following secretagogue stimulation of pancreatic acinar- (LENNARD and SINGH 1991, 1992; SINGH and WISDOM 1995) and @-cells (GYLFE 1990), in fibroblasts treated with growth factors (ISHIJIMA et al. 1991; ISHIJIMA and TATIBANA 1994), in renal epithelial cells in response to parathyroid hormone and calcito- nin (DM and QLJAMME 1992) and in vascular smooth muscle cells stimulated with endothelin and vasopressin ( O K ” et al. 1992). While these studies clearly indicate that [Mg2’Ii can be altered by extracellular factors, un-

Corresponding author Robin R. Preston, Department of Physiology, MCP-Hahnemann School of Medicine, Allegheny University of the Health Sciences, 2900 Queen Ln., Philadelphia, PA 19129. E-mail: [email protected]

Genetics 146: 871-880 (July, 1997)

equivocal evidence showing that such changes couple recognition of external stimuli with a specific cellular response is still lacking. The reasons for this are partly technical: only recently have reliable and specific methods for monitoring [Mg“], become available, so the research field is not well developed. Also, when changes in [Mg“], have been observed, they are usually small (10-30%) and slow (over several tens of minutes), which hampers analysis of their possible consequences.

Paramecium tetraurelia offers an exception to this “small and slow” rule. Paramecium is a single-celled, motile protist with a conductance that allows relatively large amounts of Mg2+ to enter the cell within millisec- onds of activation (PRESTON 1990). The resultant current (IM,) can be recorded using conventional electrophysiological techniques and has proved to be highly selective for Mg2+ over Cazf. This specificity makes it unlikely that it is due to Mg2+ permeating a nonspecific cation channel or a Ca“ channel. In attempts to better understand the functions and molecular basis for this unique conductance, we isolated mutants lacking IMg to serve as a null control in studies of the wild type. The result was a collection of mutants named “eccentric.” Mutations in eccentric A (xn tA) prevent activation of ZMg under physiological conditions, whereas this current is reduced in xntB mutants (PRESTON and KUNC 1994a,b). Studies using xntA cells revealed that Mg2+ influx v ia IMg facilitates cell repulsion from GTP (CLARK et al. 1997) and suggest that activation of this current may trigger sensory adaptation in Paramecium (PRESTON and KUNG 1994b). While the xnt mutants have already proved indispensable in defining the roles of I&, we would like to better understand the consequences of Mgzf influx by enhancing this pathway beyond normal

872 R. R. Preston and J. A. Hammond

physiological levels. Thus, we screened for mutants that are overly sensitive to Mg2+. The result was three new lines of “Chumeleon” (Cha) mutant cells. In the present report, we detail the genetic, behavioral, and electrophysiological consequences of Cha mutation in P. tetraurelia.

MATERIALS AND METHODS

Stocks and culture conditions: These studies were con- ducted using P. tetraurelia, stock 51s and the following mutants derived from this stock: d490 Paranoiac A (Pd/Pd) (VAN HOUTEN et al. 1977), d491 fas t2 (cam”/cam”, formerlyfna/ fna) (KUNG 1971; KINK et al. 1990), d4150 Paranoiac C (PaC/ PaC) (VAN HOUTEN et al. 1977), d4152 TEA-insensitive A (teaA/teaA) (CHANG and KUNC 1976), d4-619 pantophobiac B (pntB/pntB) (HINRICHSEN et al. 1985), d4623 Dancer (Dn’/ Dn’) (HINRICHSEN et al. 1984), d4644 k-shy A (ItSA’/kSA’) (Ev- ANS and NELSON 1989), d4-645 k-shy B (ksB/ksB) (EVANS and NELSON 1989), d4-646 parunoiacF(see SAIMI and KUNC 1987), d4-647 restless (rst/rst) (RICHARD et al. 1985), d4700 eccentric A (xntA’/xntA’) (PRESTON and KUNG 1994b), pa-71 1 Chameleon (Cha’/ Cha’), pa-712 Chameleon ( Chaz/ Cha’) and pa-713 Cha- meleon ( Cha3/ Cha3). The three Cha mutants were isolated during the present studies. A trichocyst non-discharge mutation (nd6/nd6) (LEFORT-TRAN et al. 1981) was used as a genetic marker in all of the crosses described below. There is no evidence for genetic linkage between nd6and any of the mutations used here. All stocks were raised at 22-28” on either a wheat grass infusion (for behavioral and genetic testing) or on an artificial medium (to produce robust cells suitable for electrophysiological recording) inoculated with Enterobacter aerogenes as described (SONNEBORN 1970; PRESTON et al. 1990b). The only significant differences between cells grown in the two media are cell size and final density.

solutions: A l l solutions contained 1 mM CaC12, 1 mM HEPES, 0.01 mM EDTA pH 7.2. Chloride salts of barium, magnesium, nickel, potassium or sodium were added to this solution as required and at the concentrations stated. Mg2+- solution contained 5 mM MgC12 and 10 mM tetraethylammo- nium (TEA’) chloride. Na+-solution contained 10 mM NaCl. Ba2+-solution contained 6 mM BaC12. K+-solution contained 30 mM KCI. Ca2+-solution contained 10 mM TEA+. Resting solution contained 4 mM KCI.

Mutagenesis: Mutations were induced in nd6 cells using either Nmethyl-W-nitro-Nnitrosoguanidine (MNNG), as described by KUNG (1971), or with y-irradiation (TAKAHASHI et al. 1985). Cha mutant lines I and I1 were selected from MNNG treated populations, whereas line I11 was isolated from a popu- lation of irradiated cells. Following mutagenesis, cell populations were separated into eight to 16 groups, starved to induce homozygosity via autogamy, and then allowed to undergo six to eight fissions before screening.

Mutant selection: Mutants with an exaggerated behavioral response to Mg‘+ were selected using a galvanotactic tech- nique, as described (HINRLCHSEN et al. 1984; PRESTON and KUNG 1994b). Two hundred milliliters of mutagenized cells in late logarithmic growth were washed twice in resting solution, concentrated by centrifugation, and then placed in a galvanotactic trough containing Mg2+-solution. An electric field was established across the length of the trough using a stimulator. Most cells responded to Mg2+ by swimming backward and were drawn toward the anode by galvanotaxis. After -20 sec, most cells had recovered from Mg’+ stimulation and com- menced swimming forward toward the cathode. Cells with an increased sensitivity to Mg’+ continued swimming backward,

however, and these individuals were removed from the trough for single-cell cloning and analysis.

Behavioral tests Ten to 20 cells were transferred from culture medium to resting solution and left undisturbed for 15 min. Individual cells were then selected with a micropipette and ejected forcibly into Mg2+-, Na+-, Ba’+-, or K+-solution. These solutions elicited backward swimming in the wild type, the duration of which was recorded with a stopwatch. Back- ward swimming times are presented as means ( t S D ) , with the significance of differences between means being calcu- lated using a Student’s t-test. Since absolute backward swimming times can vary significantly from day to day (related to undefined variations in the culture medium), all comparisons between responses of wild-type and mutant strains were made on the same day using cells that had been raised on the same batch of culture medium. All behavioral tests were carried out at room temperature (23 ? 2”).

Electrophysiology: The techniques used to record the membrane currents of Paramecium under two-electrode voltage clamp have been described (PRESTON et al. 1992a). Mg‘+ currents were elicited from cells held at -30 mV in Mg2+- solution, while Ca” currents were evoked in cells held at -40 mV in Ca“-solution.

Ni2+ resistance: Twenty cells suspended in 10-20 p1 of culture fluid were transferred to a glass well containing l ml of a Ni“ solution. At stated times, each well was examined using a dissecting microscope and its contents scored for cell survival as described previously (PRESTON and KUNG 1994b). iC2 values were determined from plots of survival score a ainst Ni2+ concentration and represent the amount of N& required to immobilize cells following 2 hr exposure.

Genetic analyses: Standard techniques were used to estab- lish genetic relationships between different strains of Parame- cium (SONNEBORN 1970). Two homozygous strains were crossed by conjugation to yield heterozygous F, progeny. The F, were then starved to induce autogamy, producing an F2 generation that was again homozygous at all loci. Unless stated otherwise, the genotype of all putative double mutants described here was confirmed in backcrosses to the wild type. The nd6 mutation was used as a genetic marker in all crosses to ensure that cross-fertilization had occurred between two mating cells and that autogamy had been induced successfully in the F,.

RESULTS

Behavioral responses to Mg2+: When wild-type paramecia are transferred from resting solution (0 mM Mg2+) to Mg2+-solution (5 mM Mg2+), they swim backward for 10-15 sec (PRESTON and KUNC 1994b). This behavior can be attributed to membrane depolarization caused by Ca2+ and Mg2+ entering the cell via separate Ca2+- and M$+- specific conductances [ZCn(d) and 1°K’ respectively (PRESTON 1990)]. Three classes of cells with enhanced Mg2+ responses were isolated following four mutageneses. The first included mutants that overreacted to all depolarizing stimuli because they lack a repolarizing K” current (SAIMI et al. 1983; HINRICHSEN et al. 1985). One of these is described elsewhere (cam‘: LING et al. 1994). A second class of mutant similarly overreacted to most depolarizing stimuli, but this strain’s phenotype resulted from a failure of the Z&l)

to inactivate normally. Similar mutants have been described by HINRICHSEN and SAIMI (1984). A third group

A wild type

n u 25

- 20 E 15

I O 3 e

% 0, El .-

.-

6 5 0 CU

* O

Mg'+-Sensitive Paramecium Mutants

B chameleon

I I 4 I I I

0 1 2 3 4 5

Culture age (days) Culture age (days)

FIGURE 1.-Variations in Paramecium swimming behavior with culture age. (A) Starved wild-type and (B) Cha mutant cells were added to fresh culture medium to a final concentration of -20 cells/ml on day zero, and then their growth followed over successive days at 28" (0 ) . On day 1, the cells had begun to grow and samples were taken for testing in Na+-solution (O), M 2 + - solution (O) , or K+-solution (A). Backward swimming responses to these solutions were tested daily until day 4, when the culture had become nutrientdepleted and the cells were again starving. Points represent mean responses of four cells. These studies were repeated on three subsequent occasions with similar results.

of mutants showed enhanced sensitivity to Mg2+ alone and are the subject of the present studies. Three lines of Cha mutant were isolated. Whereas the wild type swam backward in MgZt for 13 sec (25 sec, n = 166), line I, the best-studied of the group, swam backward for 29 sec (?9 sec, n = 107).

Dependence of the Chu behavioral phenotype on culture age: Early testing of Cha mutant behavior suggested that the sensitivity of all three lines to Mg2+ varied with growth stage. This is shown in Figure 1. Wild-type and Cha mutant (line I) cells were tested for duration of backward swimming in Mg2', Na+, and K" during successive stages of a culture cycle. Wild-type cells from an early logarithmic growth phase culture (day 1) responded to both Mg2+ and Na+ with 6-7 sec backward swimming. The duration of these responses increased slightly as cell densities approached maxi- mum, and subsequently fell again as the culture entered late logarithmic growth phase and early stationary phase. Responses to Kf remained relatively stable during days 1-3 but then increased during day 4 (Figure 1A). Figure 1B shows variations in Cha mutant behavior with growth stage. Cha line I was originally isolated from early stationary-phase cultures (day 4: Figure 1 B) , a time during which its response to Mg2+ was increased considerably compared with that of the wild type while its response to Na+ was normal. This situation was reversed during early-logarithmic growth phase, however (day 1 ), for the mutant's Mg2+ response was now normal while Na' responsiveness was enhanced. Sensitivity to Kt did not change significantly during this time. Thus, Cha cells changed from being overly Na+ sensitive to being overly Mg2+ sensitive as the culture aged. While this reversal of phenotype may not always be as dramatic

as shown in Figure 1 (particularly with respect to Na+ behavior: see Table 2), the mutant's enhanced sensitivity to Mg2+ consistently appeared during late logarithmic growth phase and early stationary phase. This allowed us to explore the genetic relationships between Cha and other mutants of P. tetraurelia (described below).

Ca*+-dependent M 2 + current: Paramecium swims backward in Mg2+ because this cation enters and depo- larizes the cell via a Mg*+-specific current ( ZMg) . Thus, cells that lack IMg either fail to respond to Mg'+ or turn briefly (PRESTON and KUNG 1994b). To examine whether the enhanced sensitivity of Cha mutants to Mg2+ might correlate with an enhanced IMg, wild-type and mutant cells were examined under two-electrode voltage clamp. IMg can be activated by either depolarizing or hyperpolarizing steps from rest, as shown in Fig- ure 2. Step depolarization of the wild type (to +20 mV) evoked first the rapidly inactivating ZCa(d) (asterisk), followed by a slower developing I,, (Figure 2A, top left). Returning to -30 mV elicited a slow inward tail current (arrowhead) caused by Mg2+ entering the cell during deactivation of IMP. Similarly, step hyperpolarization to -100 mV activated first an inactivating Ca2+ current asterisk) followed by the slowly activating IMg. Returning to -30 mV again elicited a slow IMP tail current. The amplitude of these tails has been plotted as a function of the membrane potential at which they were activated in Figure 2B. Step depolarization and hyperpolarization of Cha cells (line I) produced similar currents (Figure 2A, right). Although tail amplitudes appeared to be increased slightly compared with the wild type (Figure 2B), these differences were not significant. Significant differences in rates of tail-current


A +20 -30 mV - 0 0 30

v m 4-1 -100

wild type chameleon - " I m 7 7 T 7 7"""-r \6 _"""""""""~""

,, *"A I

200 rnsec

FIGURE 2.-Mg"-currents in wild-type and Cha mutant cells. (A) Currents were elicited from single wild-type (left) or Cha mutant (right) cells bathed in MgZf-solution using 500-msec steps from -30 mV. Upper trace in each pair shows membrane response to a step to +20 mV, whereas steps to -100 mV were used to elicit the lower traces. Depolarization elicits first a Ca2+ current asterisk) followed by ZMg. The latter current is best appreciated as a slow inward tail current when the membrane potential is returned to -30 mV (arrowhead). Membrane hyperpolarization similarly evokes a Ca2+ current (Z,,+): asterisk), followed by a slower-activating IMg (double arrowheads). Returning to -30 mV is again associated with a slow ZMg tail current (arrowhead). IMg tail currents of both the wild-type and Cha mutant cells could be described by the sum of two exponential components. The tail elicited following step hyperpolarization of the wild type decayed with time constants of 12 and 229 msec for the fast and slow component, respectively. The tail current elicited by a similar step in a Cha mutant cell (open arrow) decayed with time constants of 10 and 382 msec, the latter being significantly slower that the equivalent wild-type tail component. (B) ZMg tail currents (Iail) were elicited using 500-msec steps under voltage clamp. The amplitudes of the resultant currents are plotted against membrane potential (V,) at which they were elicited. Points are means ? SD determinations from four wild- type (.) or seven Cha mutant (0) cells: there is no significant difference in current amplitudes between the two strains.

decay between the wild type and mutant were detected, however. These are best appreciated following steps to -90 mV and below, potentials that generate strong (and hence more easily resolved) MgZt currents. In the wild type, the tail resulting from a step to -100 mV could be described by the sum of two exponentials with time constants of 12 msec and 160 msec (22 and 63 msec, respectively, n = 4). Step hyperpolarization of Cha mutants also yielded tails that decayed biexponen- tially but, whereas the time constant of the fast component was similar to that of the wild type (1 1 ? 3 msec, n = 7), the second component was slowed significantly (330 f 140 msec, n = 7: this is easily seen in the tail marked with an open arrow in Figure 2A, right).

Mg'+-currents in well-fed and starved Cha mutant cells were compared to determine whether behavioral observations above might be explained in terms of increases in current amplitude or a slowing of decay rates as the cells depleted their food source. Tail-current amplitudes were smaller in starved cells compared with well-fed cells, perhaps reflecting decreasing cell size with starvation, but these changes were not significant. Starvation had no significant effect on tail-current decay.

Since IMP is [Ca'+Ii-dependent, its slowed deactivation in Cha cells might be indicative of an underlying defect in intracellular CaP+ homeostasis. To investigate this further, we examined the rate at which recovered from inactivation. This process is also Ca" dependent and is a sensitive indicator of [Ca"Ii in Paramecium

(BREHM et al. 1980). was activated and immediately inactivated using a 20-msec step to -10 mV. To determine the time course of recovery, a second 20-msec test step to - 10 mV was applied at various intervals (10- 140 msec) after the initial, inactivating step. The magnitude of the resultant Ca2+ current is a reflection of the number of CaZt channels that have recovered from inactivation and are available for activation by the second step. In the wild type, recovery proceeded after an initial lag of 16 msec ( 2 4 msec, n = 8) with a time course that was described by a single exponential, r,,, = 35 msec ( 5 4 msec, n = 8). Similar results were ob- tained with Cha cells: recovered with a time constant of 38 msec after a lag of 17 msec ( 2 4 msec for both values, n = 7). The effects of increasing the extent of Ca'+ influx during the conditioning step on subsequent recovery were examined also (by using a 20-msec step to f 1 0 mV), but again, no significant differences were observed between wild-type and mutant cells (wild type: T , ~ ~ = 42 5 6 msec, lag = 41 2 7 msec, n = 9; Cha: r,,, = 44 +- 11 msec, lag = 44 f 12 msec, n = 9).

Ni'+-resistance: The permeability that supports ZMg also allows Ni" into the cell. This heavy metal is highly toxic to Paramecium, so mutations that alter ZMg are expected to affect sensitivity to Ni2+ also. For example, suppression of ZMg coincidentally decreases Ni'+-sensitivity 10-fold (PRESTON and KUNG 199413). Thus, we were interested to know whether enhancing IMg was correlated with an enhanced Ni'+-sensitivity. When paramecia are exposed to Ni'+, they are immobilized rap-

Mg'+-Sensitive Paramecium Mutants 875

idly and then die. The concentration required to immobilize wild-type cells following a 2-hr exposure (iC,) was 32 2 7 p~ ( n = 12). Cha mutant cells (line I) , as sus- pected, succumbed more readily to Ni2+ (iC2 = 21 t 3 p ~ , n = 12, P < 0.001).

Genetic analysis of Cha: The three Cha mutant lines were crossed to nd6, a recessive trichocyst non-discharge mutation that is commonly used as a marker in analyses of the genetic relationships between Parame- cium behavioral mutants. The three crosses yielded similar results. While the F1 progeny displayed wild-type Na+ behavior, their response to Mg2+ was intermediate between wild-type and parental response. For example, in the cross between Cha line I and nd6, the wild-type and Cha mutant parents swam backward for 12 2 5 and 39 ? 15 sec, respectively ( n = 12), while the F1 swam backward for 24 ? 9 sec ( n = 126). Inducing autogamy in the F1 yielded four classes of F2 homozygotes that segregated in a 1:l:l:l ratio of wild-type, Cha, nd6 and non-discharging Cha cells (Table 1). This suggests that lines I, 11, and I11 result from singlesite, codominant or partially dominant mutations that are unlinked to nd6.

We next tested the genetic relationships among the three Cha mutant lines. Crossing line I to line I1 produced an F1 that overresponded to both Mg2+ and Na+. Inducing autogamy in these cells produced an F2 generation that all overresponded to Mg2+ with no wild-type cells (Table 1). The normal segregation of the nd6 marker among the F2 confirmed that cross-fertilization had occurred. Line I was also crossed to line 111. Again, the F1 exhibited full Cha mutant behavior and there were no wild-type cells among the F2 (Table 1). These results suggest that the three Cha mutations form a single complementation group.

Genetic relationship between Chu and other mutations that affect Paramecium behavior: Paramecium's swimming behavior is regulated by membrane potential that is, in turn, governed by charge movements through several distinct classes of ion channel. To date, more than 20 alleles are known to regulate the activity of these channels, mutations in several of which produce phenotypes that resemble that of Cha cells. Thus, we crossed Cha line I to a number of existing behavioral mutants to investigate the mutation's novelty. Potential interactions between products of mutant genes in these crosses were investigated using solutions that test for the normal functioning of several key ion currents in Paramecium.

As noted above, the duration of backward swimming in Mg2+ solution is related to the extent of Mg2+ influx via ZMg. Well-fed wild-type cells swam backward for 7 sec in Mg'+ and this did not change significantly upon starvation (Table 2). Well-fed Cha mutant cells showed a similar response to that of the wild type when well fed, but backward-swimming times doubled upon starvation. Cha lines I1 and lines I11 showed similar increases in

Mg2+ sensitivity following starvation (Table 2). Naf solution also causes backward swimming in the wild type, a response that is caused by Na+ influx via a Ca2+-dependent Na+ current. Both well-fed and starved wild-type cells swam backward briefly in Naf (Table 2). Well-fed mutant cells (lines I and 111) swam backward for 8 sec in Na+ but response times decreased upon starvation, as described above. Baz+ causes wild-type cells to turn repeatedly ("avoidance reactions") because it enters the cell via the ciliary Ca2+ channel and triggers repeated spiking. Cha mutants showed similar responses (Table 2). Finally, K+-solution collapses the membrane potential to yield a backward-swimming episode that is largely a reflection of Ca2+ influx via ZCa(d). Wild-type cells typically swam backward for 15-20 sec in K+-solution, as did Cha mutant cells (Table 2).

Cha was crossed first to xntA, a mutant that lacks IMg (PRESTON and KUNG 199413). The resultant F1 were overly sensitive to Mg2+, reflecting the partial dominance of Cha. Inducing autogamy in the F1 yielded an F2 generation containing wild-type, Cha, and xntA cells in an approximate 1: 1:2 ratio (Table 1). Cha;xntA double mutants failed to respond to Mg2+ (Table 2), suggesting that the preponderance of xntA mutant cells among the F2 reflected epistasis of IMg expression by xntA in the double mutants. The double mutants were interesting in that their response to Ba2+ mimicked that of Cha (brief turning: Table 2) rather than xntA.

"Paranoiacs" are mutants characterized by an enhanced sensitivity to Na+. Cha's aversion to Na+ is particularly reminiscent of the Pa mutant phenotype, so the genetic relationship between these two classes of mutant was of particular interest. The F1 resulting from a cross between Cha and PaA were overresponsive to both Mg2+ and Na+ compared with wild-type cells, reflecting the partial dominance of both Cha and PaA. The F2 segregated in an approximate 1:1:2 ratio of wild type: Cha:PuA (Table 1). The double mutant was notable in that it responded only briefly to K+ (Table 2). Cha was also crossed to PaC. The F1 again overresponded to both Mg2+ and Na+ and the F2 segregated in a 1:1:2 ratio of wild type, Cha and PaC. Cha was finally crossed to stock d4-646, a mutant that has not been studied in detail previously but is also suggested to be Pa ( p g . The F1 were weakly overresponsive to Mg2+ but showed wild-type sensitivity to Na+. Inducing autogamy yielded an F2 that comprised four classes of cells in an approximate 1:l:l:l ratio (Table 1). The Cha:puFdouble mutants swam backward in Mg2+ and Na+ for significantly longer than either parent and showed an abbreviated response to K+ (Table 2). Cha was also crossed to cam", a mutant that cannot respond to Naf because it lacks INa (SAIMI 1986). cam'' coincidentally reduces sensitivity to Mg2+ (Table 2). The F1 from this cross were weakly oversensitive to Mg2+ but their response to Naf was comparable to that of the wild type. The F2 segregated in a 1:1:2 ratio of wild type:Cha:cam". Cha;cam" double


TABLE 1

Genetic analysis of Cha

Cross " F1 phenotype F2 segregation Pd

Line I X nd6

Line I1 X nd6

Line I11 X nd6

Line I X Line I1

Line I X Line 111

Cha X xntA

Cha X PaA

Cha x PaC

Cha X paF

Cha x cam1'

Cha X Dn

Cha X h A

Cha X ksB

Cha X pntB

Cha X teaA

Cha x rst

Partial Cha

Partial Cha

Partial Cha

Cha

Cha

Partial Cha

Partial Cha, Partial Pa

Partial Cha, Partial Pa

Partial Cha

Partial Cha

Partial Cha

Partial Cha

Partial Cha

Partial Cha

Partial Cha

Partial Cha

+:nd6 83535 +:nd6 81:72 +:nd6 74:67 + : nd6 6092 +:nd6 85:'77 +:nd6 85:74 +:nd6 87:64 +:nd6 50:50 + : nd6 67:87 + : nd6 89:76 +:nd6 86:53 +:nd6 68:58 + : nd6 95:93 +:nd6 53:63 +:nd6 63:66 +:nd6 85:71

+: Cha:ndG:Cha;nd6 39:44:40:45

+: Cha:ndG:Cha; nd6 35:46:35:37

+: Cha:ndb:Cha; nd6 36:38:32:35

+: Cha 0:132 + : Cha 0: 162

+: Cha:xntA 36:39:84

+ : Cha:PaA 34:41:76

+ : Cha:PaC 27:23:50

+: Cha:paF:Cha;paF

+:Cha:cam" 35:38:92 + : CharDn 35:37:67 + : Cha:hA 25:38:63 + : Cha:ksB 44:41:103 + : Cha:pntB 40:36:63

+: Cha:teaA 37:28:64 +: Cha:rst 38:35:83

35:45:38:36

>0.8

>0.5

>0.9

>0.7

>0.8

>0.5

>0.5

>0.3

>0.8

>0.2

>0.5

>0.3

>0.5

>0.5

a The recessive trichocyst nondischarge mutation (nd6) was used as a genetic marker to ensure that cross- fertilization had occurred during conjugation. nd6 does not affect the swimming behavior of Paramecium.

6F1 and F2 generations were tested for discharge competence using a saturated solution of picric acid. Behavioral phenotypes were determined from the duration of backward swimming elicited by transferring cells from resting solution to Mg2+ and Na+ solutions. At least 4 cells from each exconjugant clone were tested behaviorally in each solution and at least three pairs from each cross were tested and carried through to the Fz.

'F2 phenotype was determined from responses in Mg2+ and Na+ solution and from ability to discharge tricocysts in a saturated picric acid solution. F2 clones were tested behaviorally on at least two occasions during different stages of the growth cycle.

dSignificance was determined using a chi-squared test assuming segregation ratios of 1:1:1:1 with 3 d.f. for crosses between Cha and nd6, for crosses between the Cha lines, and also for the cross between Cha X paF. Significance of segregation among F2 progeny from all other crosses was determined assuming a ratio of 1:1:2 and 2 d.f.

~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

mutants were more responsive to Mg2+ than the cam'' ing Cha to Dn yielded an F1 that swam backward for parent and were insensitive to Na+ (Table 2). -30 sec in Mg2+ and 20 sec in Na+ (responses to Ba2+-

Dn mutation slows the rate at which the inacti- and K+-solutions were not tested). The F2 segregated vates (HINRICHSEN and SAIMI 1984). This causes pro- in a 1:1:2 ratio of wild-type, Cha and Dn cells, with the longed backward swimming in response to any depolar- Cha;Dn double mutants being indistinguishable behav- izing stimuli, a defect that is best appreciated from Dn iorally from Dn. mutants' exaggerated response to Ba2+ and K+. Cross- K-shy mutations ( h A and ksB) are suggested to raise

Mg2+-Sensitive Paramecium Mutants

TABLE 2

Behavior of wild-type and mutant paramecia in ionic test solutions

877

Mg2+ Na*

Well-fed Starved Well-fed Starved Ba2+ K+

Wild type Line I Line I1 Line IIIb xntA PaA PaC PaF cam” Dn ksA kSB pntB teaA rst Chu;xntA Cha; PaA Cha; PaC Cha; PaF Cha; cam‘’ Cha;Dn Cha; ksA Cha; ksB Cha;pntB Cha; teaA Cha; rst

~~~ ~~

BS (7) BS (8 ) BS (11) BS (11)

AR BS (1) BS (1) BS (28) BS (1) BS (12) BS (2) BS (2) BS (36)

wh BS (5)

AR BS (2) BS (3) BS (27) BS (6) BS (5) BS (2) BS (2) BS (78)

wh BS (7)

BS (5) BS (16) BS (19) BS (27)

AR

AR BS (35) BS (1) BS (9)

wh wh

BS (52) wh

BS (1) AR

BS (3) BS (3) BS (86) BS (5) BS (5)

wh BS (4) BS (93)

wh BS (3)

BS (3)

BS (4) BS ( 8 ) BS (2) BS ( 8 ) BS (2) BS (63) BS (27) BS (47)

FS BS (10) BS (19)

BS (28) wh

BS (1) BS (4) BS (60) BS (25) BS (46)

FS BS (3) BS (21) BS (1) BS (45)

Wh AR

BS (7)

BS (4) BS (4)

AR BS (4) BS (1) BS (47) BS (24) BS (48)

FS BS (4) BS ( 8 ) BS ( 8 ) BS (45)

wh AR

BS (1) BS (84) BS (29) BS (102)

FS BS ( 8 ) BS (2) BS (9) BS (46)

wh AR

AR AR AR AR

BS (52) BS (1)

AR BS (2) BS (42) BS (70)

wh wh

BS (27) BS (1)

AR AR AR AR AR

BS (54) wh wh

BS (12) AR AR

BS (4)

BS (21) BS (16) BS (18) BS (13) BS (50) BS (15) BS (20) BS (11) BS (17) BS (60) BS (36) BS (27) BS (11) BS (21) BS (25) BS (23) BS (2) BS (14) BS (4) BS (20) BS (70) BS (17) BS (27) BS (33) BS (13) BS (19)

Table summarizes responses of cells to 5 mM Mg2+, 10 mM Na+, 6 mM Ba2+ or 30 mM K+ test solutions. Responses to Mg2+ and Na+ were examined during logarithmic growth phase (“well-fed”) and in early stationary phase when the food supply had been depleted (“starved”). Cells respond to these test solutions with either forward swimming (FS), whirling (wh: a weak reversal response), avoidance reactions ( A R , resulting in cell turning) or backward swimming (BS). Numbers in parentheses indicate mean backward swimming times. Tests were carried out on a minimum of 10 cells.

intracellular free [Ca”] (EVANS and NELSON 1989). Since ICa(d) is Ca2+-inactivated, this concentration increase reduces the number of functional Ca2+ channels at rest and thereby reduces sensitivity to depolarizing stimuli. Thus, ks mutants typically only whirl in ionic test solutions that cause the wild type to swim backward (Table 2). Cha was crossed to both mutants with similar results. The F1 were partial Cha and the F2 segregated in a 1:1:2 ratio of wild type:Cha:ks. Cha;b double mutants were indistinguishable from the ks parents.

Cha was next crossed to pntB, a mutant that overreacts to stimulation because it lacks a repolarizing Ca‘+-dependent K+ current (HINRICHSEN et al. 1985). The F1 were weakly overresponsive to Mg2+, and the F2 segregated in a 1:1:2 ratio of wild type: ChapntB. The F2 included a class of mutants that responded to Mg2+ with backward swimming times that were 50% longer than the pntB parent. While these were assumed to represent Cha;pntB double mutants, this could not be confirmed genetically. Although repeated attempts were made to backcross these clones to the wild type, they would not form stable mating pairs.

Finally, Cha was crossed to two mutants that respond weakly to test solutions because they express K+ currents that activate prematurely during excitation. teaA cells have a defect in a K+ current activated upon depolarization ( HENNESSEV and KUNG 1987), whereas rst cells have a defect in a K’ current activated upon hyperpolarization (RICWARD et al. 1985; PRESTON et al. 1990a). Crossing Cha to either mutant yielded similar results. The F1 from both crosses were partial Chu, while the F2 segregated in a 1:1:2 ratio of wild type: Cha:teuA or rst. Cha;teaA double mutants could not be distinguished from tea4 in terms of responses to our test solutions: both whirled in Mg2+ and Na’. Similarly, Cha;rst con- structs behaved much like the rst parent.

DISCUSSION

Cha cells are a new class of Paramecium behavioral mutant that overreact to Mg2+. Genetic evidence suggests that the three mutants define a single complementation group and that the Cha gene is distinct from several other loci that control behavior in P. tetraurelia.


Cha mutant swimming behavior: As noted above, Par- amecium swimming speed and direction is determined by membrane potential. If the membrane is depolar- ized, the cell slows or swims backward, with the strength of ciliary reversal reflecting the magnitude of depolarization. Changes in membrane potential are coupled to ciliary activity through Ca2+. Depolarization opens voltagesensitive Ca2+ channels in the ciliary membrane, causing intraciliary [Ca"] to rise. The ciliary power stroke reverses as a consequence and the cell swims backward. Rising intraciliary Ca2+ also allows ZMg to activate. This causes Mg" to flood into the cell and causes further depolarization and the resultant backward swimming episode is sustained for as long as the Ca2+ and Mg'+ currents remain active. Forward swimming resumes only when the Ca2+ current inactivates, out- ward K' currents repolarize the membrane, and transporters restore [Ca2+Ii and [Mg2+Ii to resting levels. Thus, the Cha mutants' enhanced sensitivity to Mg2+ could reflect a defect in JMg, a defect in the ciliary Ca" current, in the repolarizing K+ currents, or in intracellular Ca2+ homeostasis.

Data shown in Table 2 begin to narrow these possibili- ties. Backward swimming in K+-solution is largely a reflection of Ca" influx via &,@). Cha cells show a wild- type response to K+, suggesting that activates and inactivates normally in these mutants. The duration of backward swimming in Ba2+-solution is a reflection of both ZCa(d) and of the repolarizing K+ currents. Thus, the loss of a Ca2+-dependent K' current in pntB cells is enough to cause prolonged backward swimming in Ba2+ (HINRICHSEN et al. 1985: Table 2 ) . Wild-type cells typically only "dance" in Ba2+ (repeated turning: Table 2 ) as do Cha cells, suggesting that membrane repolariza- tion mechanisms are intact in the mutants. Instead, the effect of Cha mutation on swimming behavior appears to be restricted to increased sensitivity to Mg2+ and, to a lesser extent, Na'.

Effects of Chu mutation on the Mg2' current: Inspec- tion of wild-type and Cha cells under voltage clamp revealed that IMP deactivates (turns off) significantly more slowly in the mutant (Figure 2). This defect would readily account for the mutant's aversion to Mg2+, for by delaying closure of the Mg2+ influx pathway, the mutation prolongs both membrane depolarization and ciliary reversal.

Since IMP is Ca2+-dependent, a delay in this current's deactivation might be indicative of a defect in intracellular Ca" regulation rather than in the Mg2+ conductance per se. The enhanced Na+ responsiveness of Cha cells would support this idea because INa is also [ Ca2+l i-

dependent. The Na+ current varies considerably from cell to cell and its deactivation kinetics are particularly sensitive to microelectrode-induced cell trauma. This makes comparing Naf tail currents between two cell strains difficult and was not attempted here. As an alternative, we examined the effects of Cha on the rate at

which IC,@) recovers from inactivation. zCa(d) inactivation results from the same rise in [Cg+], that activates ZMg and I,,, while the time course of recovery from inactivation parallels Ca2+ clearance from the cytosol (BREHM et al. 1980). We found no differences in recovery rates between wild-type and Cha cells, however, suggesting either that intracellular C g + is handled normally in Cha cells, or that if Cha mutation does affect [Ca2+Ii, it does so in a way that is not detected by this electrophysiologcal assay (see below).

Possible causes of Cha changing its behavioral "col- ors": As cell cultures age, many aspects of Paramecium physiology change also. For example, cells lose their ability to mate upon starvation and there are changes in the number and species of proteins expressed (AD- O U m E et al. 1980), in the lipid composition of the plasma membrane (KANESHIRO 1987), in cell Na+ con- tent (HANSMA 1979), and in chemosensitivity (PRESTON 1983). Thus, it is perhaps not surprising that sensitivity to ionic stimuli should change also. Presumably, these trends are a response to falling nutrient levels or a build-up of waste products.

Figure 1 shows that the sensitivity of Cha mutants to Mg'+ increases markedly as the culture approaches stationary phase. This might suggest that the mutant gene product is expressed only during late growth phase, yet the I,, deactivation defect that we think is responsible for the mutant's enhanced Mg2+ sensitivity is apparent even in well-fed cells. A second possibility is that by enhancing I&, Cha mutation simply accentu- ates a natural tendency for prolonged backward swimming in Mg2+ as the culture ages, a trend that is veiled by normal variations in backward swimming durations in the wild type. If so, mutations that enhance Mg2+- sensitivity independently of Cha might be expected to exhibit similar behavior and, indeed, paFand pntB cells both show increased sensitivity to Mg'+ as the culture ages (Table 2: differences between well-fed and starved behaviors are significant, P < 0.05). The underlying causes of this culture-dependent sensitivity may be varied and subtle and are beyond the experimental scope of this report. For example, they might reflect changes in the activity of Ca2+ and/or K' channels in response to a changing lipid environment, or changes in the efficacy of ion transport mechanisms as nutrient re- serves are depleted.

Relationship between Cha and other behavioral mutations: Genetic analyses (Table 1) suggest that Cha is a locus that has not been described previously in P. tetraurelia. There are strong resemblances between Cha mutant cells and Pa, however, mutants that are distinguished by their aversion to Na+. To date, six Pa alleles have been identified (PaA through PaF: VAN HOUTEN et al. 1977; SAIMI and KUNC 1987; BYRNE et al. 1988). PaA and PaC have been well characterized genetically and behaviorally (VAN HOUTEN et al. 1977). Their behavioral defect is specific for Na+ (Table 2) and results

M$'-Sensitive Paramecium Mutants 879

from an increase in the magnitude of the Na+ current (SAIMI 1986). PaA and PaC mutations are codominant with the wild-type allele. Preliminary studies suggested that paFwas similar to PaA and PaCin terms of genotype and phenotype (SAIMI and KUNG 1987), but subsequent work has shown paF mutations to be recessive and to enhance responsiveness to many ionic stimuli (PRESTON and KUNG 1994b; Table 2). Data in Table 1 indicate that Cha is distinct from these three Pa mutants. PaE shows close linkage to cam" (BYRNE et al. 1981). Cha shows no such linkage (Table l) , however, making it unlikely that the two loci are related. PUB was reported to be a leaky, recessive mutation (VAN HOUTEN et al. 1977), whereas PaB resulted from a dominant mutation whose responses to Na+ were increased minimally compared with the wild type. PaD had no effect on sensitivity to Mg2+ (PRESTON and KUNG 1994b). These two Pa mutants are no longer maintained in culture and hence were unavailable for genetic analysis, but reports of their phenotypes suggest that they are unlikely to be allelic with Cha.

Pathways affected by Cha mutation: A major reason for searching for Cha, as with other behavioral mutations, was to highlight molecular components of pathways that control Paramecium behavior. Ultimately, this will facilitate molecular cloning of the genes that encode ion channels and channel regulatory factors in this ciliate. While it is not possible to identify the site of lesion in Chn cells from existing data, it is useful to speculate about the possible causes of the mutants' phenotype because it helps guide investigations into the physiology of membrane excitation in Paramecium.

One plausible explanation for the Cha mutant phenotype is that, as suggested above, the Cha+ product helps regulate [Ca2+Ii. Although we have presented data ar- guing against this idea, there is growing evidence that Ca2+ fluxes occur within discrete compartments within Paramecium. For example, is known to be associated with the ciliary membrane (DUNLAP 1977), posi- tioned in such a way that Ca2+ influx via this pathway provides the trigger for ciliary reversal. A second Ca2+ conductance, &(h) , is triggered by membrane hyperpolarization (PRESTON et al. 1992a), yet the resultant Ca*+ influx occurs without causing ciliary reversal. This suggests that I&,) must be located in a compartment that is distinct from or distant from Also, while in- jecting cells with Ca2+-chelators strongly interferes with

inactivation (BREHM et al. 1980), there is little effect on Cazfdependent inactivation of &h) (PRESTON et al. 199213). Again, this suggests that the two conductances are spatially segregated. An understanding of Paramecium morphology suggests how this might be achieved. Lying immediately below the somatic portion of the plasma membrane is an extensive system of membrane-bound "alveolar" sacs. The outer alveolar membrane lies close to the somatic membrane, creating a compartment -20 nm wide that encloses its inner face

and separates it from the cytoplasm. It is the somatic portion of the plasma membrane that contains the ion channels that we record as M$+ and Na+ currents in the whole cell (SAIMI and MARTINAC 1989; SAIMI et al. 1994). If Cha+ were to encode a component of a Caz+ pump that regulates [Ca2'] within this compartment, for example, it might explain how a defect in this pump might slow ZMg deactivation while having little effect on Zc:a(d), for the latter is located in a separate (ciliary) compartment.

An alternative explanation for the Cha phenotype might be that the mutation interferes with a pathway that coregulates IMP and ZNa. Many classes of ion channel are regulated through phosphorylation state (LEVITAN 1988), so Cha+ might encode a kinase or phosphatase. While IMg has yet to be characterized fully at the single- channel level, a Ca2+-dependent Na+ channel has been described (SAIMI and LING 1990) whose activity appears to be dependent on phosphorylation state (R. R. PRES TON, unpublished observations). Coregulation of IMg and IN, would also explain how a mutation that sup- presses ZMg could also reduce I N a (xn tA: PRESTON and KUNG 1994a) and why some mutations that suppress INa coincidentally reduce ZMg (cam": KUNC et al. 1992, Table 2).

Regardless of the site of the lesion, we now have a mutation that, by exaggerating Mg2+ influx via ZMg, can direct a molecular spotlight on intracellular pathways that might be regulated by [Mg"] . Further, by selecting for mutations that suppress the Cha mutant phenotype, we may be able to identify genes that are involved in intracellular Mg2' homeostasis. While much is known about Mgz+ transporters in bacteria (MAGUIRE 1992; SMITH and MAGUIRE 1993), there is scant information about the mechanisms that maintain [Mg"] in eukary- otes. Paramecium thus offers a unique opportunity to further our understanding of the role of this important cation in eukaryotic cell activity.

We are grateful to Dr. CHINC KUNC (University of Wisconsin-Madi- son) for providing many of the behavioral mutants used in this study and to the National Institutes of Health for their support (GM-51498).

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a Paramecium Mutant With an Enhanced Sensitivity to Magnesium

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