Vincent A , Lautermilch NJ , Spitzer NC .

NS07220 NINDS NIH HHS

	Fig. 1. Antisense (AS) RNA to xKv3.1specifically inhibits the current expressed in oocytes injected withxKv3.1 mRNA. Currents were recorded after injection of cRNA encoding xKv genes ± xKv3.1AS. Currents recorded in the presence of AS were normalized to those recorded from oocytes of the same clutch in the absence of AS.A, xKv3.1 AS was injected at a stoichiometry of 4:1 with xKv3.1 RNA. AS decreased the current by 86% without shifting the I–V relation.B–D, xKv3.1 AS had no significant effect on currents encoded by xKv1.1, xKv2.1, orxKv4.3 when mRNA was injected at the same stoichiometry.Circles, xKv RNA alone;squares, xKv RNA plusxKv3.1 AS. n ≥5 for each gene.
	Fig. 2. Inhibition of expression of xKv3.1transcripts by antisense RNA injection is visualized by in situ hybridization of whole-mount Xenopus embryos with a xKv3.1-specific antisense probe. A, Sense (S) probe gives no signal in uninjected control embryos. B, AS probe reveals that endogenous xKv3.1 mRNA is present in the spinal cord of control embryos at stage 22 and thereafter (Gurantz et al., 2000).C, S probe labeling of AS-injected embryos demonstrates stability of injected xKv3.1 AS. Signal is observed in 16-cell through stage 30 embryos; dorsal view of another embryo indicates that only the injected side of the embryo (top) contains AS. D, Injection of xKv3.1 AS reduces the endogenous signal detected with the AS probe. Dorsal view of another embryo indicates that reduction of the in situ hybridization signal is restricted to the injected side (top, arrowheads). Area inbox is enlarged at right. For all embryos anterior is to the right and dorsal isup. Scale bar, 1 mm. Black spots on tailbud embryos are melanocytes.
	Fig. 3. Inhibition of expression ofI Kv in cultured neurons after injection ofxKv3.1 AS RNA into a single blastomere of two-cell stage embryos. FITC-dextran was coinjected to identify neurons containing AS. Whole-cell currents elicited from a holding potential of −80 mV and steps from −40 to +60 mV reveal two subpopulations of neurons. One population, F AS* (fluorescent AS-injected, affected) neurons, has reducedI Kv amplitude and activation kinetics. The other, F AS (fluorescent AS-injected, unaffected) neurons, exhibits currents that are not different from those of nonfluorescent (NF) controls. The records are from three neurons at 18–22 hr in culture.
	Fig. 4. Properties of I Kv in FAS* neurons. Currents were recorded at 18–22 hr in culture. A, Current densities of FAS*neurons are reduced relative to those of NF and FASneurons. FAS* cells show a mean 62 ± 1% decrease in current density at 0 to +60 mV (p < 0.01), whereas FAS cells have currents that are not different from NF controls. The boxes include all measurements from cells at a particular voltage. Horizontal bars indicate the means for these measurements, and vertical barsindicate SEMs. NF, Nonfluorescent;F AS, fluorescent, but unaffected by AS;F AS*, fluorescent, affected by AS. Values were designated FAS* when they exceeded 2 SD from the mean values for NF neurons in the same culture dishand thus in some cases overlap the values for NF neurons recorded in other cultures; n = 27. B, Times to half-activation (t 1/2max) of FAS* neurons are increased relative to those of NF and FAS neurons. FAS* cells show a 66 ± 6% increase in t 1/2max at +20 to +60 mV (p < 0.01), whereas FAS cells have currents that are not different from NF controls. Values at 0 mV are not shown because I Kv is small and slowly activating at this potential in FAS* neurons, and discrimination from noise is ambiguous; n = 27. Noise precluded measurement from two cells at +20 mV.
	Fig. 5. Injection of xKv3.1 sense RNA into a single blastomere of two-cell stage embryos has no effect on the expression of I Kv in cultured neurons.A, Current densities of FS and NF neurons are not different. B, Times to half-activation of FS and NF neurons are not different. Conventions are as in Figure 4; n = 18.
	Fig. 6. xKv3.1 AS increases action potential duration. The AP of FAS* neurons is longer in duration than that of NF and FAS neurons (p < 0.01), consistent with reduction of the amplitude of I Kv. The records are from three neurons at 1 d in culture; n = 24.
	Fig. 7. The percentage of neurons in whichI Kv and APs are affected byxKv3.1 AS is similar to the percentage of neurons previously shown to express xKv3.1 transcripts (Gurantz et al., 2000). Numbers in parenthesesindicate the number of neurons affected/tested for each condition.
	Fig. 8. I Ca is not affected by the expression of xKv3.1 AS. A, Whole-cell voltage-clamp records of low voltage-activated and high voltage-activated calcium currents; potassium and sodium currents were suppressed pharmacologically. The records are from two neurons at 1 d in culture. Cells were held at −80 mV and stepped to potentials from −50 to +20 mV. B, Current densities for NF and FAS neurons. The records are from six and seven neurons at 1 d in culture. Conventions are as in Figure4. Mean I Ca is not significantly different for these two populations. The apparent reduction in the density of calcium current at −10 mV likely reflects outliers in the NF population.
	Fig. 9. Computer-simulated action potentials in whichI Kv is reduced by the amounts observed experimentally are longer in duration, consistent with the increase in experimentally recorded AP duration after the suppression ofI Kv with xKv3.1 AS.A, Computer simulations ofI Kv in control and experimental conditions (100% I Kv, and with reduction in density and increase in time to half-maximal activation values derived from AS experiments). B, Simulations ofI Ca in control and experimental conditions; the jagged nature of the traces is attributable to the plotting regime, which increases the distance between points when the rate of change of the current is small. C, Simulations of APs in control and experimental conditions. Modeling was performed by using a previously developed program (Lockery and Spitzer; 1992; Gu and Spitzer, 1993). Mimicking experimental recording conditions, the holding potential was set at −60 mV, and brief 2.5 msec depolarizing current pulses were used to elicit APs arising after the termination of the stimulus.
	kcnc1 (potassium channel, voltage gated Shaw related subfamily C, member 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 29 and 30, lateral view, anterior right, dorsal up.

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