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Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the 42 Nicotinic Acetylcholine Receptor

Division of Biology, California Institute of Technology, Pasadena, California (N.O.R.-P., H.A.L., B.N.C.); Luxtera, Inc., Carlsbad, California (T.J.P.); and Molecular Devices Corp., Union City, California (A.F.)

Received January 18, 2005; accepted May 17, 2005

	Abstract

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Extracellular Ca²⁺ robustly potentiates the acetylcholine response of 42 nicotinic receptors. Rat orthologs of five mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)—4(S252F), 4(S256L), 4(+L264), 2(V262L), and 2(V262M)—reduced 2 mM Ca²⁺ potentiation of the 42 1 mM acetylcholine response by 55 to 74%. To determine whether altered allosteric Ca²⁺ activation or enhanced Ca²⁺ block caused this reduction, we coexpressed the rat ADNFLE mutations with an 4 N-terminal mutation, 4(E180Q), that abolished 42 allosteric Ca²⁺ activation. In each case, Ca²⁺ inhibition of the double mutants was less than that expected from a Ca²⁺ blocking mechanism. In fact, the effects of Ca²⁺ on the ADNFLE mutations near the intracellular end of the M2 region—4(S252F) and 4(S256L)—were consistent with a straightforward allosteric mechanism. In contrast, the effects of Ca²⁺ on the ADNFLE mutations near the extracellular end of the M2 region—4(+L264)2, 2(V262L), and 2(V262M)—were consistent with a mixed mechanism involving both altered allosteric activation and enhanced block. However, the effects of 2 mM Ca²⁺ on the 42, 4(+L264)2, and 42(V262L) single-channel conductances, the effects of membrane potential on the 2(V262L)-mediated reduction in Ca²⁺ potentiation, and the effects of eliminating the negative charges in the extracellular ring on this reduction failed to provide any direct evidence of mutant-enhanced Ca²⁺ block. Moreover, analyses of the 42, 4(S256L), and 4(+L264) Ca²⁺ concentration-potentiation relations suggested that the ADNFLE mutations reduce Ca²⁺ potentiation of the 42 acetylcholine response by altering allosteric activation rather than by enhancing block.

At physiological concentrations (1.5-2 mM), Ca²⁺ potentiates the neuronal nicotinic agonist response by binding to an N-terminal site in the receptor protein (Galzi et al., 1996; Le Novere et al., 2002; Rodrigues-Pinguet et al., 2003). Allosteric Ca²⁺ activation of the neuronal nicotinic receptors is conserved across species (rat, human, chicken) and across nicotinic receptor subtypes (Mulle et al., 1992; Vernino et al., 1992; Galzi et al., 1996; Steinlein et al., 1997; Liu and Berg, 1999). Adding 2 to 2.5 mM Ca²⁺ to the extracellular saline increases the wild-type 42 acetylcholine response by 300 to 500% (Steinlein et al., 1997; Figl et al., 1998; Rodrigues-Pinguet et al., 2003). A common feature of the ADNFLE mutations is that they reduce 2 to 2.5 mM Ca²⁺ potentiation of the 42 acetylcholine response by 50 to 72%, at acetylcholine concentrations 30 µM (Steinlein et al., 1997; Figl et al., 1998; Rodrigues-Pinguet et al., 2003). Because the extracellular Ca²⁺ concentration in the mammalian brain is normally 1.5 to 2 mM (Egelman and Montague, 1999), a decrease in Ca²⁺ potentiation of the 42 acetylcholine response could contribute to ADNFLE seizures either by (1) reducing 42-mediated inhibitory transmitter release in the cortex (McNamara, 1999) or (2) shifting the balance between 42-mediated excitatory and inhibitory transmitter release during bouts of high-frequency cortical synaptic activity, in favor of excitatory transmitter release (Rodrigues-Pinguet et al., 2003).

At concentrations outside the physiological range (20 mM), Ca²⁺ also reduces the single-channel conductance of the 42 receptor (Buisson et al., 1996). The ability of Ca²⁺ to both block and potentiate 42 nicotinic receptors means that at least two distinct molecular mechanisms could account for the effects of the ADNFLE mutations on Ca²⁺ potentiation. The mutations could 1) interfere with allosteric Ca²⁺ activation or 2) enhance the potency of uncompetitive or noncompetitive Ca²⁺ block. There are several classes of possible Ca²⁺ blocking mechanisms. We refer to them simply as "noncompetitive."

The 4 N-terminal mutation 4(E180Q) eliminates allosteric Ca²⁺ activation of the 42 receptor (Rodrigues-Pinguet et al., 2003). To decide whether the ADNFLE mutations reduce Ca²⁺ potentiation by interfering with allosteric Ca²⁺ activation or by enhancing Ca²⁺ block, we constructed double-mutant receptors containing this mutation and one of five rat orthologs—4(S252F), 4(S256L), 4(+L264), 2(V262L), and 2(V262M)—of the human ADNFLE mutations—4(S248F), 4(S252L), 4(776ins3), 2(V287L), and 2(V287M). We refer to these receptors as 4(E180Q):ADNFLE double mutants. The rat 4(+L264) mutation is a 3-base pair insertion that adds a leucine at position 264 to the 4 amino acid sequence.

If the Ca²⁺ blocking mechanism is correct, then the percentages by which Ca²⁺ blocks the acetylcholine response of 4(E180Q):ADNFLE double-mutant receptors and by which the corresponding single ADNFLE mutations reduce Ca²⁺ potentiation of the wild-type acetylcholine response should be the same. On the other hand, if the mutations reduce Ca²⁺ potentiation of the acetylcholine response by altering allosteric Ca²⁺ activation, then the 4(E180Q) mutation should nullify the effects of the ADNFLE mutations, and Ca²⁺ should not block the 4(E180Q):ADNFLE double-mutant acetylcholine response any more than it blocks 4(E180Q)2 acetylcholine response. We also measured the effects of the 4(S256L) and 4(+L264) mutations on the Ca²⁺ concentration-potentiation relation for the acetylcholine response, the effects of 2 mM Ca²⁺ on the wild-type, 4(+L264)2, and 42(V262L) single-channel conductance, and the voltage dependence of the 2(V262L)-mediated reduction in Ca²⁺ potentiation. Finally, we determined whether eliminating the fixed negative charge in the extracellular ring at the extracellular pore entrance could reverse the effects of the ADNFLE mutations on Ca²⁺ potentiation. The results show that the ADNFLE mutations reduce Ca²⁺ potentiation of the 42 acetylcholine response by disrupting allosteric Ca²⁺ activation of the receptor rather than by enhancing Ca²⁺ block.

	Materials and Methods

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Oocyte Expression. Stage V to VI Xenopus laevis oocytes were surgically isolated as described previously (Quick and Lester, 1994). Female X. laevis frogs were anesthetized by immersion in 0.2% tricaine methanesulfonate (Sigma, St. Louis, MO), pH 7.4, for 45 to 60 min, and the ovarian lobes were extracted through a small abdominal incision. The oocyte follicular layer was removed using Type A collagenase (1-2 h in a 2 mg/ml collagenase solution; Sigma). To increase nicotinic receptor expression, the rat 4-1 (Goldman et al., 1987) and 2 inserts (Deneris et al., 1988) were subcloned into a vector containing a 5'-untranslated region from the alfalfa mosaic virus that enhanced protein translation and a long 3' poly A tail (Figl et al., 1998). We used the QuikChange single and multiple site-directed mutagenesis kit (Stratagene, La Jolla, CA) to construct the rat 4 and 2 mutations and verified them by DNA sequencing. Capped cRNA was synthesized in vitro using the mMessage mMachine RNA transcription kit (Ambion, Austin, TX). After a 24-h incubation in a modified Barth's solution containing 96 mM NaCl, 5 mM HEPES, 2.5 mM sodium pyruvate, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 0.6 mM theophylline (Sigma) with 2.5 µg/ml Gentamicin (Sigma) and 5% horse serum, pH 7.4 (Irvine Scientific, Santa Ana, CA), the isolated oocytes were injected with rat 4 and 2 cRNA.

Whole-Oocyte Electrophysiology. Injected oocytes were incubated for 24 h in the modified Barth's solution at 15-19°C before electrophysiological recordings were attempted. We voltage-clamped the oocytes with two 3-M, KCl-filled microelectodes (1.5- to 4-M resistance) using a GeneClamp 500 voltage clamp (Axon Instruments, Union City, CA). During the voltage-clamp recordings, the oocytes were continually superfused with a nominally Ca²⁺-free saline (ND98) containing 98 mM NaCl, 1 mM MgCl₂, and 5 mM HEPES, pH 7.4, at 20-23°C, unless otherwise stated. We added 0.1 to 2 mM CaCl₂ to the ND98 solution to measure Ca²⁺-induced changes in the acetylcholine response. Acetylcholine was applied to the oocytes using a U-tube microperfusion system (Cohen et al., 1995). The time constant for solution exchange was 0.5 s. The voltage-clamp currents were digitized using a personal computer equipped with a DigiData 1322A analog-to-digital interface and pCLAMP version 8 software (Axon Instruments). To avoid aliasing, the voltage-clamp currents were filtered at one fourth to one fifth of the sampling frequency (typically 20 Hz) with an 8-pole, low-pass Bessel filter. We measured the current-voltage relation of the acetylcholine-induced current by ramping the membrane potential between -120 and +50 mV over a 0.5-s interval, in the presence and absence of 1 µM acetylcholine, and digitally subtracting the ramp current in the presence of acetylcholine from that in the absence of acetylcholine. We used Dunnett's test (SigmaStat ver. 1; SPSS Inc., Chicago, IL) to determine whether the ADNFLE mutations significantly affected Ca²⁺ potentiation of the peak acetylcholine response. The errors reported in the text are S.E.M. or S.E. (for fitted parameters). We used a previous approximation for the variance of the ratio of two random variables (Mood et al., 1974) to calculate the S.E.M. for the mutant-induced fractional reductions in Ca²⁺ potentiation of the acetylcholine response.

Desensitization Analysis. To compare desensitization of the wild-type and mutant responses, we fit their desensitizing phases to the sum of an exponential component and a constant term using the curvefitting routine in pCLAMP version 8.2. From these fits, we obtained an apparent time constant of desensitization (_D) and the percentage of steady-state desensitization of the response (SSD), defined as

where I_exp and I_C are the fitted amplitudes of the exponential component and constant, respectively. We analyzed the effects of the mutations and Ca²⁺ on these two parameters using a two-way analysis of variance with receptor type and Ca²⁺ concentration as factors. Post hoc comparisons were carried out on the ranked data using the Student-Newman-Keuls test.

BAPTA Injections. To prevent Ca²⁺ influx through the wild-type and mutant nicotinic receptors from activating endogenous Ca²⁺-activated Cl^- currents, we injected the oocytes with 50 nl of a 100 mM K₄BAPTA solution buffered to pH 7.4 with 10 mM HEPES, 5 to 10 min before recording from them (Haghighi and Cooper, 2000; Rodrigues-Pinguet et al., 2003). Assuming an oocyte volume of 500 nl, these injections produced a final intracellular BAPTA concentration of 10 mM.

View larger version (22K): [in this window] [in a new window]   Fig. 1. The 4(E180Q) mutation abolishes 2 mM Ca2+ potentiation of the rat wild-type (WT) 42 1 mM acetylcholine (ACh) response. A, location of the 4(E180Q) mutation in the 4 N-terminal domain (top). The linear map above the aligned sequences shows the major domains of the nicotinic subunit and the location of loops A to F in the N terminus. The aligned amino acid sequences (bottom) show that the Glu at 4(E180) is conserved across different species (rat, human, chick) and neuronal subunits. Bold letters denote the positions of the 4(E180Q) and 2(E177Q) mutations. Identical residues in the wild-type subunits are highlighted in light gray. B-D, voltage-clamped responses of the wild-type, 4(E180Q)2, and 42(E177Q) receptors to 1 mM acetylcholine in 0 and 2 mM added extracellular Ca2+. The lines above the traces show the acetylcholine applications. V = -50 mV. E, the bars show the Ca2+-induced changes in the peak wild-type, 4(E180Q)2, and 42(E177Q) responses. The error bars are ± S.E.M. (n = 4-8 oocytes). Asterisks denote significant differences from the wild-type (P < 0.05).

	Results

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The 4(E180Q) Mutation Abolishes Ca²⁺ Potentiation of the 42 Acetylcholine Response. To ensure that the 4(E180Q) mutation eliminated allosteric Ca²⁺ activation of the 42 receptor, we measured its effects on 2 mM Ca²⁺ potentiation of the 42 1 mM acetylcholine response. We used 1 mM acetylcholine for these experiments instead of the 30 µM concentration used previously (Rodrigues-Pinguet et al., 2003) to maximize any potential open-channel Ca²⁺ block of the ADNFLE mutant receptors and to minimize any effects that mutant-induced shifts in acetylcholine potency might have on Ca²⁺ potentiation of the acetylcholine response. Adding 2 mM Ca²⁺ to a nominally Ca²⁺-free saline (ND98; see Materials and Methods) increased the peak 1 mM wild-type acetylcholine response by 310 ± 30% (mean ± S.E.M.) at -50 mV (Fig. 1B; Table 1). In contrast, adding 2 mM Ca²⁺ reduced the 4(E180Q)2 1 mM acetylcholine response by 10 ± 1% (Fig. 1, C and E; Table 1). Thus, consistent with previous experiments using 30 µM acetylcholine (Rodrigues-Pinguet et al., 2003), the 4(E180Q) mutation completely abolished 2 mM Ca²⁺ potentiation of the 42 1 mM acetylcholine response (Fig. 1; Table 1). It is interesting that a 2 mutation—2(E177Q)—homologous to the 4(E180Q) mutation did not reduce 42 Ca²⁺ potentiation (Fig. 1, D and E; Table 1).

The 4(S252F) and 4(S256L) Mutations Obviously Alter Allosteric Ca²⁺ Activation. To determine whether the ADNFLE mutations reduced Ca²⁺ potentiation by enhancing Ca²⁺ block of the receptor or by altering allosteric Ca²⁺ activation, we compared the effects of 2 mM Ca²⁺ on the ADNFLE single- and 4(E180Q):ADNFLE double-mutant acetylcholine responses. Ca²⁺ affected the double mutants with ADNFLE mutations closer to the intracellular end of the M2 region—4(E180Q:S252F)2 and 4(E180Q: S256L)2—differently from those with ADNFLE mutations near the extracellular end of M2—4(E180Q:+L264)2, 4(E180Q)2(V262M), and 4(E180Q)2(V262L). We shall discuss the results for the ADNFLE mutations closer to the intracellular end first. Consistent with their previously reported effects on Ca²⁺ potentiation of the 30 µM acetylcholine response (Rodrigues-Pinguet et al., 2003), the single 4(S252F) and 4(S256L) mutations significantly reduced 2 mM Ca²⁺ potentiation of the 42 1 mM acetylcholine response (Fig. 2, A and B; Table 1). Adding 2 mM Ca²⁺ increased the 4(S256L)2 and 4(S252F)2 1 mM acetylcholine responses by only 40% (Fig. 2, A, B, and E) as opposed to a 310% increase for the wild-type (Fig. 1, B and E). Thus, the Ca²⁺-induced mutant increases were 60% less than that of the wild-type. If enhanced Ca²⁺ block accounted entirely for the effects of the 4(S252F) and 4(S256L) mutations on Ca²⁺ potentiation, then 2 mM Ca²⁺ should have also reduced the 4(E180Q:S252F)2 and 4(E180Q:S256L)2 1 mM acetylcholine responses by 60%. However, 2 mM Ca²⁺ had little effect on the 4(E180Q:S252F)2 and 4(E180Q: S256L)2 responses (Fig. 2, C, D, and F; Table 1). In fact, it reduced the 4(E180Q:S252F)2 1 mM acetylcholine response by only 10 ± 1%, and it increased the 4(E180Q: S256L)2 1 mM acetylcholine response by 10 ± 4%. Therefore, the 4(S252F) and 4(S256L) mutations seem to reduce 42 Ca²⁺ potentiation by diminishing allosteric Ca²⁺ activation of the 42 receptor rather than by enhancing Ca²⁺ block.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Combining the 4(S252F) or 4(S256L) mutation with the 4(E180Q) mutation does not enhance 2 mM Ca2+ inhibition of the 4(E180Q)2 acetylcholine response. A-D, voltage-clamped 1 mM acetylcholine responses of the 4(S256L)2, 4(S252F)2, 4(E180Q:S256L)2, and 4(E180Q: S252F)2 receptors in 0 and 2 mM added extracellular Ca2+. V = -50 mV. E and F, the bars show the Ca2+-induced changes in the peak wild-type, 4(S256L)2, 4(S252F)2, 4(E180Q)2, 4(E180Q:S256L)2, and 4(E180Q:S252F)2 responses. The error bars are ± S.E.M. (n = 4-8 oocytes). Asterisks denote significant (P < 0.05) differences from the wild-type (E) or 4(E180Q)2 receptors (F).

The Effects of the 4(+L264), 2(V262M), and 2(V262L) Mutations Are More Complex. The single 4(+L264), 2(V262M), and 2(V262L) ADNFLE mutations reduced Ca²⁺ potentiation of the acetylcholine response by an amount similar to that of the 4(S252F) and 4(S256L) mutations (Fig. 3, A, C, E, and G; Table 1) but Ca²⁺ inhibited the 4(E180Q:+L264)2, 4(E180Q)2(V262M), and 4(E180Q)2(V262L) double-mutant receptors more than the 4(E180Q)2 receptor. Adding 2 mM Ca²⁺ to the extracellular solution increased the 4(+L264)2 1 mM acetylcholine response by only 10 ± 2%, it increased the 42(V262M) response by only 40 ± 4%, and it actually reduced the 42(V262L) response by 20 ± 4% (Fig. 3G and Table 1). Thus, similar to 4(S252F) and 4(S256L) mutations, the 4(+L264), 2(V262M), and 2(V262L) mutations reduced 2 mM Ca²⁺ potentiation of the acetylcholine response by 55 to 75% compared with the wild-type. However, in contrast to the 4(S252F) and 4(S256L) mutations, adding 2 mM Ca²⁺ reduced the 4(E180Q:+L264)2, 4(E180Q)2(V262M), and 4(E180Q)2(V262L) double-mutant 1 mM acetylcholine responses by 30 to 50% (Fig. 3, B, D, F, and H; Table 1). Thus, Ca²⁺ inhibited the 4(E180Q:+L264)2, 4(E180Q)-2(V262M), and 4(E180Q)2(V262L) responses significantly (P < 0.05) more than the 4(E180Q)2 response (Fig. 3H; Table 1). Nevertheless, Ca²⁺ inhibition of the double-mutant responses was less than that predicted from the corresponding single-mutant reductions in Ca²⁺ potentiation by a pure blocking mechanism (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Combining the 4(+L264), 2(V262L), or 2(V262M) mutation with the 4(E180Q) mutation does enhance 2 mM Ca2+ inhibition of the 4(E180Q)2 acetylcholine response. A-F, voltage-clamped 1 mM acetylcholine responses of the 4(+L264)2, 42(V262L), 42(V262M), 4(E180Q:+L264)2, 4(E180Q)2(V262L), and 4(E180Q)2(V262M) receptors in 0 and 2 mM added extracellular Ca2+. V = -50 mV. G and H, the bars show the Ca2+-induced change in the peak wild-type, 4(+L264)2, 42(V262L), 42(V262M), 4(E180Q)2, 4(E180Q:+L264)2, 4(E180Q)2(V262L), and 4(E180Q)2(V262M) responses. The error bars are ± S.E.M. (n = 4-6 oocytes). Asterisks denote significant (P < 0.05) differences from the wild-type (G) or 4(E180Q)2 receptors (H).

View larger version (17K): [in this window] [in a new window]   Fig. 4. Ca2+ inhibition of the E180Q:ADNFLE double-mutant acetylcholine responses is less than that expected from a noncompetitive Ca2+ blocking model. Y axis, the percentage inhibition of the peak E180Q: ADNFLE double-mutant 1 mM acetylcholine response by 2 mM added extracellular Ca2+ (Observed Inhibition). X axis, the percentage by which the corresponding single ADNFLE mutations reduced 2 mM Ca2+ potentiation of the wild-type 1 mM acetylcholine response (Predicted Inhibition). The dashed line has a slope of unity and denotes equal observed and predicted inhibition. The horizontal and vertical error bars are ± S.E.M. (n = 4-8 oocytes).

The 4(+L264) and 2(V262L) mutations did not significantly affect the peak amplitude of 1 mM acetylcholine response in 0 mM Ca²⁺ (P > 0.05). However, the 2(V262M) mutation significantly (P < 0.05) increased it. The peak amplitudes of the 42(V262M), 4(+L264)2, and 42(V262L) 1 mM acetylcholine responses in 0 mM Ca²⁺ were 4000 ± 2000 (n = 5), 3000 ± 2000 (n = 4), and 500 ± 240 nA (n = 4), respectively. Thus, the 42, 4(+L264), 2(V262L), and (V262M) mutations did not reduce the amplitude of the 1 mM acetylcholine response in 0 mM Ca²⁺.

Altered Allosteric Ca²⁺ Activation Explains the Effects of the ADNFLE Mutations on the Ca²⁺ Concentration-Potentiation Relation Better than Enhanced Ca²⁺ Block. There are two possible interpretations of the effects of Ca²⁺ on the 4(E180Q:+L264)2, 4(E180Q)2(V262M), and 4(E180Q)2(V262L) acetylcholine responses. First, the 4(+L264), 2(V262M), and 2(V262L) mutations could reduce 42 Ca²⁺ potentiation by both enhancing Ca²⁺ block and inhibiting allosteric Ca²⁺ activation of the receptor. Second, the 4(E180Q) mutation could interact with these particular ADNFLE mutations to convert the allosteric Ca²⁺ binding site from a positive to a negative site. To decide between these two alternatives, we compared the effects of two of the ADNFLE mutations on the Ca²⁺ concentration-potentiation relation and asked whether they were better explained by a mutant-induced change in allosteric Ca²⁺ activation of the receptor or enhanced Ca²⁺ block. We chose the 4(S256L) mutation as representative of the ADNFLE mutations closer to the intracellular end of M2 and, the 4(+L264) mutation as representative of those closer to the extracellular end. We used 30 µM acetylcholine for these experiments rather than 1 mM to avoid cumulative receptor desensitization by repeated acetylcholine applications.

Consistent with a small or negligible Ca²⁺ block of the wild-type receptor between 0.1 and 2 mM Ca²⁺, a simple hyperbolic binding function (eq. 1) fit the wild-type data well (R² = 0.98; Fig. 5, A and B):

(2)

where the I_Ca/I₀ is the ratio of the peak 30 µM acetylcholine response with added extracellular Ca²⁺ to that in 0 mM added Ca²⁺, the [Ca²⁺]_o is the added extracellular Ca²⁺ concentration, the EC₅₀ is the half-maximal [Ca²⁺]_o for potentiation of the acetylcholine response, and the P_max is the maximum value of the I_Ca/I₀. The fitted wild-type P_max and EC₅₀ were 3.6 ± 0.1 and 0.4 ± 0.1 mM (df = 6), respectively. To determine whether enhanced Ca²⁺ block could account for the effects of the ADNFLE mutations on the Ca²⁺ concentration-potentiation relation, we multiplied the wild-type hyperbolic binding function (eq. 1) by a simple inhibitory binding isotherm and fit this function (eq. 2) to the 4(S256L)2 and 4(+L264)2Ca²⁺ concentration-potentiation relations (Fig. 5, A and B):

(3)

where the IC₅₀ is the half-maximal inhibitory concentration for Ca²⁺ block of the mutant receptor and the expression,

describes allosteric Ca²⁺ activation of the receptor (assuming that it is unaffected by the mutations). This function fit the 4(S256L)2 and 4(+L264)2 Ca²⁺ concentration-potentiation data poorly (R² = 0; Fig. 5, A and B) because it predicted a relief from Ca²⁺ block; thus, a relief from the mutant-mediated reductions in Ca²⁺ potentiation as the [Ca²⁺]_o approached zero. The fitted IC₅₀ values for Ca²⁺ block of the 4(S256L)2 and 4(+L264)2 receptors were 0.8 ± 0.3 mM (df = 4) and 1.27 ± 0.03 mM (df = 4), respectively. Thus, enhanced Ca²⁺ block cannot account for the effects of the 4(S256L) and 4(+L264) mutations on the 42 Ca²⁺ concentration-potentiation relation.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Altered allosteric Ca2+ activation accounts for the effects of the 4(S256L) and 4(+L264) mutations on the Ca2+ concentration-potentiation relation better than enhanced Ca2+ block. A and B, fits of the wild-type Ca2+ concentration-potentiation data to a hyperbolic binding function (eq. 1 under Results) and the 4(S256L)2 and 4(+L264)2 Ca2+ concentration-potentiation data to the product of this hyperbolic function and a Ca2+ inhibitory isotherm (eq. 2 under Results). C and D, fits of the wild-type, 4(S256L)2, and 4(+L264)2 data to an allosteric Ca2+ activation model (eq. 3 under Results). Eqs. 1 to 3 were fit to the data using the nonlinear least-squares regression routine implemented in SigmaPlot version 8 (SPSS, Inc.). See the text for the fitted parameter values. The error bars are ± S.E.M. (n = 3-8 oocytes). V = -50 mV.

(5)

where the K_Ca is the equilibrium constant for Ca²⁺ binding to the open receptor state (see the online Supplement for the derivation of eq. 3). Because the parameters K_Ca and P_max were highly covariant, it was appropriate to fix the P_max beforehand and to let only the K_Ca vary during the fit. To fit eq. 3 to the wild-type data, we fixed the P_max at 3.6 (estimated from fitting eq. 1 to the wild-type data). With this constraint, eq. 3 fit the wild-type data as well as eq. 1 (R² = 0.98; Fig. 5, C and D) and yielded a K_Ca of 108 ± 3 µM (df = 7). Visual inspection of the 4(S256L)2 and 4(+L264)2 data indicated that both mutations reduced the P_max for Ca²⁺ potentation (Fig. 5, C and D). Fixing the P_max at 1.6 gave reasonable fits to the 4(S256L)2 and 4(+L264)2 data (R² = 0.75, 0.56, respectively; Fig. 5, C and D) and yielded K_Ca values of 1.2 ± 0.4 mM and 200 ± 100 µM (df = 4), respectively. Thus, a mutant-induced change in allosteric Ca²⁺ activation accounts for the effects of ADNFLE mutations on the Ca²⁺ concentration-potentiation relation better than enhanced Ca²⁺ block. Therefore, an interaction between the ADNFLE mutations closer to the extracellular end of M2 and the 4(E180Q) mutation that creates a negative Ca²⁺ allosteric site may account for their more complex effects.

The ADNFLE Mutations Enhance Steady-State De-sensitization in 2 mM Ca²⁺. In the absence of any added extracellular Ca²⁺, previous results show that the five ADNFLE mutations we studied do not consistently affect acetylcholine-induced desensitization (Figl et al., 1998; Rodrigues-Pinguet et al., 2003). However, we only tested the effects of three of the mutations—4(S256L), 2(V262L), and 2(V262L)—in 2 mM Ca²⁺ previously. In 2 mM Ca²⁺, these three mutations increase steady-state desensitization of the 30 µM acetylcholine response (Rodrigues-Pinguet et al., 2003). The effects of the 4(S252F) and 4(+L264) mutations on desensitization in 2 mM Ca²⁺ have not previously been reported. To determine whether all five mutations affect desensitization in 2 mM Ca²⁺, we analyzed desensitization of the 1 mM acetylcholine responses obtained in the Ca²⁺ potentiation experiments above. The desensitizing phase of these responses primarily reflects the fast component of de-sensitization because we only applied acetylcholine for 5 to 16 s to minimize cumulative receptor desensitization by repeated agonist applications. The time course of desensitization of these responses was adequately fit by the sum of a single exponential component and a constant term (Fig. 6, A and B). As in previous studies (Figl et al., 1998; Rodrigues-Pinguet et al., 2003), the ADNFLE mutations did not consistently affect the SSD or _D of desensitization (see Materials and Methods) in 0 mM Ca²⁺. However, all five mutations significantly (P < 0.05) increased the SSD in 2 mM Ca²⁺ (Fig. 6C; Table 2). In 2 mM Ca²⁺, the SSD of the mutant receptors was 38 to 77% larger than that of the wild-type. Thus, the ADNFLE mutations increased steady-state desensitization in 2 mM Ca²⁺. The mutations did not consistently affect the _D in 0 or 2 mM Ca²⁺ (Fig. 6D; Table 2).

View larger version (30K): [in this window] [in a new window]   Fig. 6. The ADNFLE mutations enhance the SSD of the acetylcholine response in 2 mM Ca2+. A and B, fits of the desensitizing phases of the wild-type (A) and 4(+L264)2 (B) 1 mM acetylcholine responses in 0 and 2 mM Ca2+ to the sum of an exponential component and a constant term. The vertical dashed lines show the extent of the fitted region. The fits are superimposed on the traces over this region. C and D, the bars show the SSDs (C) and desensitization time constants (D) (D) of the wild-type and ADNFLE mutant 1 mM acetylcholine responses in 0 and 2 mM Ca2+. The error bars are ± S.E.M. (n = 4-8 oocytes). Asterisks denote a significant (P < 0.05) difference from the wild-type receptor. The crosses denote a significant effect of Ca2+ on the SSD (C) or D (D).

View this table: [in this window] [in a new window]   TABLE 2 Percent steady-state desensitization (SSD) and desensitization time constant (D) of the 1 mM acetylcholine responses in 0 and 2 mM Ca2+ The SSD and D values are presented as means ± S.E.M.

Consistent with previous results (Rodrigues-Pinguet et al., 2003), Ca²⁺ did not affect the SSD of the wild-type 1 mM acetylcholine response. However, it significantly (P < 0.05) reduced the _D by 51% (Fig. 6, C and D; Table 2). Thus, Ca²⁺ increased the speed, but not the depth, of fast wild-type desensitization. It is interesting, the 4(E180Q) mutation eliminated this effect (Table 2).

Combining the 4(E180Q) and ADNFLE mutations only significantly affected desensitization of the 42(V262M) and 4(+L264)2 responses (Table 2). Combination with the 4(E180Q) mutation significantly (P < 0.05) reduced the SSD of the 42(V262M) response in 0 and 2 mM Ca²⁺ and brought the mutant SSDs closer to the wild-type values (Table 2). It also significantly (P < 0.05) increased the _D in 2 mM Ca²⁺ but it did not eliminate the effects of Ca²⁺ on the mutant _D (Table 2). Thus, the 4(E180Q) mutation reduced steady-state desensitization of the 42(V262M) response in both 0 and 2 mM Ca²⁺ and, reduced the effects of Ca²⁺ on its time course of desensitization. Combining the 4(E180Q) with the 4(+L264) mutation significantly (P < 0.05) reduced the 4(+L264)2 SSD in 0 mM Ca²⁺ but, did not significantly affect the SSD in 2 mM Ca²⁺ or, the _D in 0 or 2 mM Ca²⁺ (Table 2). Thus, the 4(E180Q) mutation did not consistently affect desensitization of the ADNFLE mutant responses.

The 2(V262L)-Induced Reduction in Ca²⁺ Potentiation Is Voltage-Independent. Another way to test the hypothesis that ADNFLE mutations near the extracellular end of M2 enhance Ca²⁺ block of the 42 channel is to examine the voltage dependence of their effects on Ca²⁺ potentiation. If the mutations enhance Ca²⁺ block at a site within the membrane electric field, then their effects on Ca²⁺ potentiation should be voltage-dependent. To measure the effects of the 2(V262L) mutation on Ca²⁺ potentiation at positive (+30 mV) and negative (-50 mV) membrane potentials, we used a second mutation—4(E245Q)—expected to relieve inward rectification of the 42 acetylcholine response. Severe inward rectification of the wild-type acetylcholine response precludes accurate measurements of Ca²⁺ potentiation at positive membrane potentials. To overcome this limitation, we used the 4(E245Q) mutation. Similar to the previously reported 4(E245A) mutation (Haghighi and Cooper, 2000), the 4(E245Q) mutation relieved inward rectification of the 42 acetylcholine-induced current-voltage relation (n = 5; Fig. 7A), allowing us to easily measure responses at positive potentials. It is interesting that the 4(E245Q) mutation also significantly reduced 2 mM Ca²⁺ potentiation of the 42 1 mM acetylcholine response at -50 mV (P < 0.05; Fig. 7B), but its effects were less severe than the ADNFLE mutations (Table 1). Ca²⁺ increased the 4(E245Q)2 1 mM acetylcholine response by 90 ± 30% (n = 5) at -50 mV (Fig. 7B) and by 80 ± 20% (n = 10) at +30 mV (Fig. 7, D and F). Thus, consistent with previous results for native neuronal nicotinic receptors (Amador and Dani, 1995), Ca²⁺ potentiation of the 4(E245Q)2 acetylcholine response was voltage-independent. Coexpression of the 4(E245Q) and 2(V262L) mutations yielded a double-mutant receptor that responded robustly to 1 mM acetylcholine at both negative and positive membrane potentials (Fig. 7, C, E, and F). If the 2(V262L) mutation reduces Ca²⁺ potentiation by enhancing Ca²⁺ block at a site within the membrane electric field, then positive membrane potentials should relieve the block and thus relieve the 2(V262L)-mediated reduction in 4(E245Q)2 Ca²⁺ potentiation. However, the Ca²⁺-induced change in the 4(E245Q)2(V262L) acetylcholine response at -50 mV (-50 ± 10%, n = 5) was not significantly (P > 0.05) different from that at +30 mV (-30 ± 10%, n = 13) (Fig. 7, C, E, and F), consistent with previous results showing that the 4(S252F) and 4(+L264) ADNFLE mutations similarly affect Ca²⁺ potentiation at membrane potentials of -100 and -50 mV (Figl et al., 1998). Thus, enhanced Ca²⁺ block of the 4(E245Q)2(V262L) receptor at a site inside the membrane electric field cannot account for the reduced Ca²⁺ potentiation of this receptor.

View larger version (18K): [in this window] [in a new window]   Fig. 7. The 2(V262L) mutation reduces Ca2+ potentiation of the 4(E245Q)2 response almost equally well at negative (-50 mV) and positive (+30 mV) membrane potentials. A, acetylcholine-induced current-voltage relations for the wild-type and 4(E245Q)2 receptors in 0 mM added Ca2+, generated by ramping the membrane potential from -120 to +50 mV. To facilitate comparison, the 4(E245Q)2 ACh-induced current was scaled up to match the value of the wild-type current at -120 mV. The wild-type and mutant current-voltage relations are from individual oocytes and represent typical results for five oocytes in each case. [acetylcholine] = 1 µM. B and C, voltage-clamped 1 mM ACh responses of the 4(E245Q)2 and 4(E245Q)2(V262L) receptors in 0 and 2 mM added extracellular Ca2+ at -50 mV. D and E, records at +30 mV. F, the bars show the Ca2+-induced changes in the 4(E245Q)2 and 4(E145Q)2(V262L) responses at -50 and +30 mV. The error bars are ± S.E.M. (n = 5-13 oocytes).

Eliminating Negative Charges in the Extracellular Ring Does Not Prevent the 2(V262L) Mutation from Reducing Ca²⁺ Potentiation.

View larger version (18K): [in this window] [in a new window]   Fig. 8. The 4(E266Q) mutation eliminates the fixed negative charge in the extracellular ring of the 42 receptor but does not relieve the effects of the 2(V262L) mutation on 2 mM Ca2+ potentiation. A and B, voltage-clamped 1 mM acetylcholine responses of the 4(E266Q)2 and 4(E266Q)2(V262L) receptors in 0 and 2 mM added extracellular Ca2+. V = -50 mV. C, The bars show the Ca2+-induced changes in the 4(E266Q)2 and 4(E266Q)2(V262L) responses. The error bars are ± S.E.M. (n = 4 oocytes).

View larger version (38K): [in this window] [in a new window]   Fig. 9. Adding 2 mM Ca2+ to the pipette-filling solution (98 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.4) significantly (P < 0.01) reduces the wild-type, but not the 4(+L264)2 and 42(V262L), single-channel current in cell-attached patches. A, large (L) and small (S) wild-type single-channel currents in 0 mM added extracellular Ca2+. The solid line is the mean baseline (closed-channel) current (0 pA). The dotted lines show the mean current amplitudes of the large and small wild-type openings. The values of the mean amplitudes are given in parentheses. B, all-points amplitude histograms for the single-channel currents in A. The ordinate is the number of points (# Events). The abscissa is the current (I) in picoamperes. The smooth lines are fits to the sum of two Gaussian components. Letters denote the mean baseline (B), large-amplitude (L), and small-amplitude (S) currents. C to D, wild-type single-channel currents in 2 mM added extracellular Ca2+ and their amplitude histograms. E and F, the data for the 42(V262L) single-channel currents in 0 mM added extracellular Ca2+. G and H, the data for the 42(V262L) single-channel currents in 2 mM added extracellular Ca2+. I and J, 4(+L264)2 single-channel currents in 0 and 2 mM added extracellular Ca2+ and their amplitude histograms. The pipette voltage was +100 mV (driving potential of -100 mV).

Previous results show that 10 to 20 mM added extracellular Ca²⁺ reduces the conductance of neuronal nicotinic channels (Vernino et al., 1992; Amador and Dani, 1995; Buisson et al., 1996). Adding 2 mM Ca²⁺ to the pipette-filling solution reduced the i_s values for the two wild-type states by a small, but significant (P < 0.01), amount (Fig. 9, A-D; Table 3). It reduced the small i_s by 19 ± 5% and the large wild-type i_s by 9 ± 2%. The g_s for the large wild-type conductance state in 2 mM Ca²⁺ (40 ± 4 pS, n = 6) (Table 3) was similar to that reported previously (46 pS) for the predominant conductance state of human 42 nicotinic receptors expressed in human embryonic kidney cells and measured in outside-out patches using 120 mM external Na⁺, 2 mM external Ca²⁺, and 120 mM internal K⁺ (Buisson et al., 1996). The addition of 2 mM Ca²⁺ also slightly reduced the i_s values for the mutant conductance states (Fig. 9, E-J; Table 3), but the reductions were not significant (P > 0.05). Thus, the 4(+L264) and 2(V262L) mutations do not enhance Ca²⁺-induced reductions in the 42 single-channel current.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Less effective Ca²⁺ potentiation of the acetylcholine response is a common feature of all the ADNFLE mutations tested so far. Consistent with previous results for the 30 µM acetylcholine response (Steinlein et al., 1997; Figl et al., 1998; Rodrigues-Pinguet et al., 2003), rat orthologs of five of the six reported ADNFLE mutations reduce 2 mM Ca²⁺ potentiation of the 1 mM acetylcholine response by 55 to 74%. The effect of the sixth ADNFLE mutation—4(T265I)—on Ca²⁺ potentiation has not been reported (Leniger et al., 2003). Contrary to previous suggestions (Rodrigues-Pinguet et al., 2003), a change in allosteric Ca²⁺ activation, rather than enhanced Ca²⁺ block, seems to be responsible for the ADNFLE mutant-induced reductions in Ca²⁺ potentiation. Several lines of evidence support this conclusion. Ca²⁺ at a concentration of 2 mM does not inhibit the 4(E180Q:S252F)2 and 4(E180Q:S256L)2 responses any more than it inhibits the 4(E180Q)2 response. The effects of the 4(S256L) and 4(+L264) mutations on the Ca²⁺ concentration-potentiation relation are more consistent with altered allosteric Ca²⁺ activation than enhanced Ca²⁺ block. The effects of the 2(V262L) mutation on 4(E245Q)2 Ca²⁺ potentiation are voltage-independent. The 4(E266Q) mutation also fails to relieve the 2(V262L)-mediated reduction in Ca²⁺ potentiation. Finally, 2 mM Ca²⁺ does not significantly reduce the 4(+L264)2 and 42(V262L) single-channel conductances.

It is interesting that the five ADNFLE mutations we tested also increased the apparent steady-state desensitization of the 1 mM acetylcholine response in 2 mM Ca²⁺. Because our acetylcholine applications lasted only 5 to 16 s, this increase could reflect either a real increase in steady-state desensitization or a decrease in the relative amplitude of the slow component of desensitization. Regardless of its origin, this effect does not seem to be a common feature of the mutations because the sixth reported ADNFLE mutation—4(T265I)— does not seem to affect desensitization of the acetylcholine response in Ca²⁺ (Leniger et al., 2003).

The effects of Ca²⁺ on the 4(E180Q:+L264)2, 4(E180Q)2(V262M), and 4(E180Q)2(V262L) double-mutant responses allow two possible interpretations. The 4(+L264), 2(V262M), and 2(V262L) mutations could reduce Ca²⁺ potentiation of the acetylcholine response by a mixed mechanism involving both altered allosteric Ca²⁺ activation and enhanced Ca²⁺ block. On the other hand, these mutations could interact with the 4(E180Q) mutation to change the allosteric Ca²⁺ site from a positive to a negative one. The effects of the 4(+L264) mutation on the Ca²⁺ concentration-potentiation relation, and the failure of 2 mM Ca²⁺ to reduce the 4(+L264)2 and 42(V262L) single-channel conductance more than the wild-type, support the latter interpretation. The voltage independence of the 2(V262L)-mediated reduction in 4(E245Q)2 Ca²⁺ potentiation also shows that the 2(V262L) mutation does not enhance Ca²⁺ block at a site within the membrane electric field, and the failure of the 4(E266Q) mutation to relieve the 2(V262L)-mediated reduction in Ca²⁺ potentiation shows that the 2(V262L) mutation does not enhance Ca²⁺ block at negatively charged residues in the extracellular ring.

Consistent with previous results (Galzi et al., 1996; Rodrigues-Pinguet et al., 2003), the 4(E180Q) mutation abolishes Ca²⁺ potentiation of the 42 1 mM acetylcholine response. The homologous 2 mutation—2(E177Q)— does not. The results for both mutations agree with a previous model of the 42 allosteric Ca²⁺ binding site (Le Novere et al., 2002) in which the Glu at 4(E180), but not that at 2(E177), directly contributes to Ca²⁺ binding. However, if combining the 4(E180Q) mutation with the ADNFLE mutations near the extracellular end of M2 produces a negative allosteric Ca²⁺ binding site, then the 4(E180Q) mutation cannot prevent Ca²⁺ binding to the 42 receptor.

Similar to 7 nicotinic receptors (Galzi et al., 1996), 2 mM Ca²⁺ increases the maximum 42 acetylcholine response 3-fold. If we assume that 1) Ca²⁺ does not amplify the neuronal nicotinic acetylcholine response by removing receptor inactivation (Amador and Dani, 1995) and 2) the ADNFLE mutations do not enhance Ca²⁺ block of the receptor, then two mechanisms could account for the effects of the ADNFLE mutations on the Ca²⁺-induced increase in acetylcholine efficacy. First, the ADNFLE mutations could increase acetylcholine efficacy in the absence of Ca²⁺ (decrease K₂ in Scheme 1; see the online Supplement), thereby reducing the number of available receptors that could be opened by adding extracellular Ca²⁺. Second, the mutations could reduce the Ca²⁺ affinities of the closed, agonist-bound (K ₃^-1 in Scheme 1; see the online Supplement) and/or open 42 receptors (mK₃^-1 in Scheme 1; see the online Supplement), thereby reducing the ability of Ca²⁺ to increase acetylcholine efficacy. Because the rat 4(S252F) and 4(+L264) ADNFLE mutations do not affect surface antibody binding or increase the maximum (500 µM) acetylcholine response in 0 mM added Ca²⁺ (Figl et al., 1998), these mutations at least do not seem to reduce Ca²⁺ potentiation by increasing acetylcholine efficacy in the absence of Ca²⁺. Comparable data are not available for the 4(S256L), 2(V262M), and 2(V262L) mutations. However, our results show that adding 2 mM Ca²⁺ reduces the 42(V262L) 1 mM acetylcholine response by 20 ± 4%. At most, increasing the acetylcholine efficacy in the absence of Ca²⁺ could eliminate Ca²⁺ potentiation. It could not produce a Ca²⁺-induced decrease in the maximum acetylcholine response, as observed for the 2(V262L) mutation. Thus, this mutation also does not seem to reduce Ca²⁺ potentiation by increasing acetylcholine efficacy in 0 mM added Ca²⁺. However, we cannot rule out this mechanism for the 4(S256L) and 2(V262M) mutations.

Previous results show that three rat ADNFLE mutations— 4(S256L), 2(V262M), and 2(V262L)—reduce 42 Ca²⁺ potentiation in an acetylcholine concentration-dependent manner (Rodrigues-Pinguet et al., 2003). These mutations significantly reduce Ca²⁺ potentiation of the 30 µM and 1 mM acetylcholine responses but not that at lower acetylcholine concentrations (10 or 50 nM). In contrast, the 4(S252F) and 4(+L264) mutations reduce Ca²⁺ potentiation of the 10 and 50 nM and 30 µM acetylcholine responses equally well. Although uncompetitive Ca²⁺ inhibition, at a site independent of the one mediating allosteric Ca²⁺ activation, could explain the acetylcholine concentration dependence of the 4(S256L), 2(V262M), and 2(V262L) effects on Ca²⁺ potentiation, the evidence above argues against this explanation. An alternative explanation is receptor heterogeneity. There are least two distinct 42 subtypes with different pharmacological properties and subunit stoichiometries (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Nelson et al., 2003; Zhou et al., 2003). The 4(S256L), 2(V262M), and 2(V262L) mutations may selectively reduce Ca²⁺ potentiation of the low-affinity, but not the high-affinity, 42 subtypes. A third possible explanation, at least for the effects of the 4(S256L) and 2(V262M) mutations, is that these mutations reduce Ca²⁺ potentiation by increasing acetylcholine efficacy in the absence of Ca²⁺. Such an increase would not affect Ca²⁺ potentiation at the foot of the acetylcholine concentration-potentiation relation (where a large fraction of additional receptors are available for opening), but it would reduce Ca²⁺ potentiation at higher acetylcholine concentrations, where a smaller fraction of additional receptors is available for opening.

Mechanism of Seizure Generation. The effects of the ADNFLE mutations on Ca²⁺ potentiation could facilitate seizure generation in two ways. If the mutations do not affect surface receptor expression or acetylcholine efficacy in 0 mM Ca²⁺, then their effects on Ca²⁺ potentiation should reduce 42-mediated inhibitory release in vivo. This loss could facilitate seizure generation by reducing 42-mediated lateral inhibition in the cortex (McNamara, 1999). The rat 4(S252F) and 4(+L264) mutations do not affect surface anti-4 or -2 antibody binding and do not increase the 500 µM acetylcholine response in the absence of added Ca²⁺ (Figl et al., 1998). Thus, these two mutations could reduce the peak acetylcholine response under physiological Ca²⁺ concentrations. Consistent with this prediction, the human 4(S248F), 4(S252L), and 2(V287M) mutations—orthologous to the rat 4(S252F), 4(S256L), and 2(V262M) mutations—reduce the maximum acetylcholine response in 2.5 mM added extracellular Ca²⁺ by 50% (Bertrand et al., 1998; Bertrand et al., 2002). However, these three mutations do not reduce the maximum acetylcholine response of mock heterozygous receptors (created by injecting X. laevis oocytes with a mixture of wild-type and mutant cRNA) (Bertrand et al., 2002), and ADNFLE patients are heterozygous for the mutant alleles. Cellular mRNA and protein regulation could also affect ADNFLE receptor expression in vivo. Hence, determining whether the ADNFLE mutations reduce the 42 acetylcholine response in vivo will ultimately require measurements in knock-in mice.

The ADNFLE mutant-mediated reductions in Ca²⁺ potentiation could also alter the balance between 42-mediated excitatory and inhibitory transmitter release during bouts of synchronized, high-frequency firing in the cortex (Rodrigues-Pinguet et al., 2003). Presynaptic nicotinic receptors facilitate both excitatory and inhibitory neurotransmitter release in the central nervous system (Wonnacott, 1997; Alkondon et al., 2000). Because of the limited extracellular space in the brain, synchronous repetitive firing may selectively deplete Ca²⁺ from the extracellular space around excitatory synapses (Vassilev et al., 1997; Egelman and Montague, 1999; Rusakov and Fine, 2003). Ca²⁺ regulation of the neuronal nicotinic agonist response may act as a negative feedback mechanism to down-regulate nicotinic receptor-mediated excitatory transmitter release during high-frequency synaptic firing (Amador and Dani, 1995). In general, Ca²⁺ depletion at excitatory synapses may protect against 42-initiated seizures by ensuring that 42-mediated inhibitory transmitter dominates 42-mediated excitatory transmitter release during bouts of synchronized, high-frequency cortical firing, such as sleep spindles. Reducing 42 Ca²⁺ potentiation could impair the ability of 42-mediated lateral inhibition to contain the spread of 42-mediated excitation in the cortex and thus allow 42-mediated excitation to initiate a seizure.

	Footnotes

 
This work was supported by grants from the National Institute of Health (NS0438000 and NS011756, to B.N.C. and H.A.L., respectively and a minority supplement (NS0438000-04S1 to N.O.R.).

Portions of this work were published previously by UMI publishers in a doctoral dissertation by N.O.R.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.011155.

ABBREVIATIONS: ADNFLE, autosomal dominant nocturnal frontal lobe epilepsy; [Ca²⁺]_o, added extracellular Ca²⁺ concentration; i_s, single-channel current; g_s, single-channel conductance; SSD, percentage of steady-state desensitization; _D, time constant of desensitization.

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

Address correspondence to: Bruce N. Cohen, Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125. E-mail: bncohen{at}caltech.edu

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