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Figure 2. Phosphorylation of Kv3.4 NH2-terminal peptides by three PKC isoforms. Diacylglycerol-stimulated phosphorylation of the wild-type peptide and the mutant peptide S[8,9,15,21]A by PKCα, and PKCβ, and PKCγ (materials and methods). Values are expressed as the mean ± SD (n = 3). |
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Figure 3. Comparison of PKC action and the effect of NH2-terminal S → D mutations on Kv3.4 inactivation. From a holding potential of −100 mV, 900-ms depolarizing pulses to +50 mV evoked all the currents. The solid horizontal line indicates the zero current level. (top) Currents expressed by wild type, S[15,21]A, and S[8,9]A in the absence and presence of 20 nM PMA (see Fig. 1). Currents are shown without normalization. (bottom) Currents expressed by S → D mutants (S[8,9,15,21)D, S[8,9]D, and S[15,21]D) and their corresponding wild type (no PMA was applied). For comparison, wild-type and S → D mutant peak currents are shown normalized. |
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Figure 4. The rates of rapid inactivation of wild-type (WT) Kv3.4 and NH2-terminal S → D mutants. (A) Whole-oocyte currents evoked by 112-ms depolarizing pulses to +50 mV from a holding potential of −100 mV. Capacitive transients were subtracted using a scaled smooth template with no active currents (materials and methods). Each pair compares a mutant (thick trace) current and its corresponding wild type from the same batch of oocytes (thin trace). The solid horizontal line indicates the zero current level. Rates of rapid inactivation were determined as described in materials and methods. For mutant channels that exhibited very slow inactivation, long pulses (900 ms) were used to estimate the time constants of current decay. (B) Tukey box plot summarizing the data from wild type (WT) and mutants. Data were ordered from fast (top) to slow (bottom). Left and right borders of a box represent the 25th and 75th percentiles, respectively. Solid and dashed lines in the box correspond to the 50th percentile and the mean value, respectively. Left and right whiskers represent the 10th and 90th percentiles, respectively. Outliers are shown as •. The number of oocytes examined is indicated by the figure accompanying the boxes. Four oocytes expressing S[8,9,15]D were also examined. However, the time constants of inactivation from this mutant were not determined because it exhibited no inactivation in response to 900-ms pulses to positive membrane potentials. Open symbols with a dot represent the predicted rate of inactivation for multiple mutants assuming a simple additive effect of single mutations as described in materials and methods. |
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Figure 5. The rate of recovery from inactivation of wild-type (WT) Kv3.4 and several S → D mutants. (A) Time courses of recovery from inactivation of S8D, S9D, S15D, and S21D (○) and their corresponding wild-type from the same batch of oocytes (•). A two-pulse protocol was used to determine the time course of recovery from inactivation at −100 mV. Whole-oocyte currents were evoked and inactivated by a 150-ms step depolarization to +40 mV (p1) from a holding potential of −100 mV. This pulse inactivated ≥80% of the currents expressed by wild type, S8D, S9D, S15D, and S21D. The degree of inactivation at the end of the pulse decreases for the mutants that disrupted inactivation to a greater extent (∼20% for S[8,15,21]D). To allow recovery from inactivation, the interpulse interval (IPI, at −100 mV) was gradually incremented; and a 75-ms test pulse to +40 mV (p2) evaluated the recovery. The peak of the current evoked by the test pulse was divided by the peak of the current evoked by the first pulse (Ip2/Ip1), and this ratio was plotted against the interpulse interval. The time course of recovery was described assuming the sum of two exponential terms (solid lines, see text). The relative weight of the fast term was >65%. (B) Tukey box plot summarizing the results of the analysis of the fast rate of recovery from inactivation. Definitions of this plot are as described herein. The number of oocytes examined is indicated by the figure accompanying the boxes. We also examined S[8,9,15]D, S[8,9,21]D, and S[8,9,15,21]D. However, they were not analyzed quantitatively because they exhibited little or no inactivation (<20% using a 900-ms pulse) or the shortest interpulse interval used in these experiments (50–100 ms) allowed complete recovery from inactivation. Open symbols with a dot represent the predicted rate of recovery from inactivation for multiple mutants assuming a simple additive effect of single mutations as described in materials and methods. |
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Figure 6. Prepulse inactivation of wild-type Kv3.4 and several S → D mutants. (A) Graph of the normalized peak current against prepulse potential. During a 10-s period, the membrane was held at the indicated prepulse potentials (abscissa). A 112-ms test pulse to +40 mV was delivered at the end of the prepulse. Between episodes, the membrane potential was held at −100 mV and the interepisode interval was no longer than 6 s. The peak of the current evoked by the test pulse was plotted against the prepulse potential and the resulting curve was described assuming a Boltzmann distribution (solid line) to estimate the midpoint potential (V0.5), the slope, and the fraction of noninactivating current (Table I). For display, data were normalized to the maximum current estimated from the Boltzmann distribution (I/Imax). Prepulse inactivation curves from S[8,9]D, triple mutants, and S[8,9,15,21]D were not analyzed quantitatively because they are significantly biased by current activation (notice that these curves exhibit a reduced steepness and a rebounding effect at the foot of the curves). In these cases, the symbols are connected by a dotted line. (B) Tukey box plot summarizing the midpoint potentials of the prepulse inactivation curves from wild-type and several double S → D mutants (see Fig. 4, legend, for definitions). The number of oocytes examined is indicated by the figure accompanying the boxes. Open symbols with a dot represent the predicted values for the double mutants assuming a simple additive effect of single mutations (relative to wild type, the shifts in the midpoints were added and the result added to the wild-type value). |
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Figure 7. The effects of neutral and negatively charged substitutions affecting S8. (A) Whole-cell currents from oocytes expressing wild-type Kv3.4 (WT) and the mutants S8N, S8D, and S8E. The pulse protocol is shown above the currents. For comparison, currents are shown normalized. The peak currents were (μA): 8.5, 10.4, 3.5, and 10.9 for wild type, S8N, S8D, and S8E, respectively. Capacitive transients were subtracted on-line using a P/4 protocol. The solid horizontal line indicates the zero current level. (B) Time courses of recovery from inactivation of Kv3.4 (WT) and the mutants S8N, S8D, and S8E. The pulse protocol and analysis were as described in Fig. 5, legend. The time course of recovery was described assuming the sum of two exponential terms (solid lines, see text). The relative weight of the fast term was >65%. (C and D) Tukey box plots summarizing the rate constants of inactivation and recovery from inactivation obtained from the experiments described in A and B. Definitions of this plot are as described in Fig. 4, legend. The number of oocytes examined is indicated by the figure accompanying the boxes. Data for wild type and S8D were replotted from Figs. 4 and 5. |
