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The effect of changing the internal pH on the maximal rate of forward glutamate uptake was tested by whole-cell voltage-clamping the cells using pipette

Figure 3.6 - Relationship between external pH, Km and Vmax

A. The Km for external glutamate (real Km from figure 3.5) plotted as a fimction of external pH.

B. The carrier’s Vmax (from figure 3.4) plotted as a function of the Km for external glutamate (from figure 3.5).

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Figure 3.7 - The sodium-dependence of the maximal glutamate uptake current at different external pH values

Whole-cell voltage-clamped Müller cells were held at -40mV. The current evoked by 200p,M glutamate is plotted as a fimction of external sodium concentration for external pH 7.3, 6.1 and 5.0. The currents are normalized to the value obtained with 1 lOmM Na^ at external pH 7.3. The data points are mean+SEM of the data from 5 cells. The data were fit with the square of a Michaelis-Menten function (inset), and the parameters o f these fits are shown on the right. An acid pH decreased the sodium affinity with little effect on the maximum current at a saturating external sodium concentration.

Solutions External solutions contained (in mM) KCl 2.5, NaCl 110, MgCE 0.5, CaCE 3, BaCE 6, glucose 15 and buffer 5. For external pH 7.3 the buffer used was HEPES, for pH 6.1 and 5.0 MES was used. The pH was adjusted to the desired value with NMDG. The sodium concentration was reduced by replacing NaCl with choline-Cl.

The internal solution contained (in mM) KCl 50, K2EGTA 5, NaCl 5, NazATP 5,

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solutions highly buffered to different pH values. Low series resistance (typically less than 4 MQ) pipettes were used, and recordings were not made until the cell membrane had been broken for at least 5 minutes. This was intended to ensure that there had been adequate dialysis of the intracellular solution. A near- saturating dose of glutamate (200p,M) was applied to determine the rate of glutamate uptake at each internal pH. Uptake currents in different cells were compared, so errors introduced by variability in the size of the cells were reduced by normalizing each cell’s current to its capacitance (Barbour et al. 1991).

Control experiments were performed to test if the different buffers used to examine a wide pH range directly affected the internal surface of the glutamate uptake carrier. Internal solutions of the same pH, but with different buffers were used. The pH was chosen to be mid-way between the pKa o f the buffers concerned, so that the buffering power of the two solutions were approximately equal. For internal pH 6.8, buffered with either MES or HEPES, the maximal glutamate uptake currents (normalized by cell capacitance) were 0.39±0.04pA/pF and 0.38±.05pA/pF (mean±SEM of data from 5 cells) respectively. There is no significant difference between the currents recorded with these two buffers (p=0.90, two-tailed t-test). For internal pH 8.0, buffered with either TAPS or HEPES, the maximal glutamate uptake currents were 0.69±0.08pA/pF and 0.74±0.08pA/pF (mean+SEM of data from 6 cells) respectively. There is no significant difference between the currents recorded with these two buffers either (p=0.68, two-tailed t-test). Thus differences in uptake current seen at different internal pH values (figure 3.8) presumably reflect the altered pH rather than the altered buffer species or concentration.

Sample currents recorded from different cells at different internal pH values are shown in figure 3.8A. The uptake current was increased at more alkaline values of internal pH. Averaged values are plotted as a function of internal pH in figure 3.8B. The data are best-fit with a Michaelis-Menten dependence of current

Figure 3.8 - Internal pH-dependence of the maximal rate of forward uptake

A. Membrane currents recorded in whole-cell voltage-clamped Müller cells at different values of internal pH. The holding potential was -40mV. The black bar above each trace represents the bath application of a near-saturating dose of glutamate (200pM). Data for each different value of internal pH were obtained from different cells clamped using pipette solutions of different pH values.

B. The magnitude of the maximal forward uptake current plotted as a function of internal pH. The currents are normalized to the cells’ capacitance. The data are fit with a Michaelis-Menten dependence on the internal hydroxide ion concentration, proportional to [OH ]i/([OH Ji+Km) with KM=43nM The points are the meantSEM of the data from at least 5 cells.

Solutions The external solution contained (in mM) KCl 2.5, NaCl 104.5, MgCb 0.5, CaCl2 3, BaCb 6, glucose 15 and HEPES 5. The pH was adjusted to

7.3 with NMDG.

Internal solutions contained (in mM) KCl 50, KzEGTA 5, NaCl 5, Na2ATP 5,

CaCb 1, MgCb 7 and buffer. For pH 7.0, 71mM HEPES was used as the buffer and the pH was adjusted to 7.0 with 25mM NMDG. For pH values between 6.8 and 7.9 the concentrations of HEPES and NMDG were adjusted appropriately. For pH values less than 6.8, HEPES was replaced by MES. For pH values above 7.9, HEPES was replaced by TAPS.

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