Kv4.2 mRNA abundance and A-type K(+) current amplitude are linearly related in basal ganglia and basal forebrain neurons - PubMed (original) (raw)

T Tkatch et al. J Neurosci. 2000.

Abstract

A-type K(+) currents are key determinants of repetitive activity and synaptic integration. Although several gene families have been shown to code for A-type channel subunits, recent studies have suggested that Kv4 family channels are the principal contributors to A-type channels in the somatodendritic membrane of mammalian brain neurons. If this hypothesis is correct, there should be a strong correlation between Kv4 family mRNA and A-type channel protein or aggregate channel currents. To test this hypothesis, quantitative single-cell reverse transcription-PCR analysis of Kv4 family mRNA was combined with voltage-clamp analysis of A-type K(+) currents in acutely isolated neurons. These studies revealed that Kv4.2 mRNA abundance was linearly related to A-type K(+) current amplitude in neostriatal medium spiny neurons and cholinergic interneurons, in globus pallidus neurons, and in basal forebrain cholinergic neurons. In contrast, there was not a significant correlation between estimates of Kv4.1 or Kv4.3 mRNA abundance and A-type K(+) current amplitudes. These results argue that Kv4.2 subunits are major constituents of somatodendritic A-type K(+) channels in these four types of neuron. In spite of this common structural feature, there were significant differences in the voltage dependence and kinetics of A-type currents in the cell types studied, suggesting that other determinants may create important functional differences between A-type K(+) currents.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

Low-threshold, TEA-resistant A-type current was present in most of the neurons tested. Left Column, Current traces evoked by the voltage-clamp protocol shown on the top in each of the cell types studied are shown. Right Column, The difference currents from the column on the left are shown (control). In addition, the difference currents recorded in the presence of 20–50 m

m

TEA are shown. Note the improved isolation afforded by the addition of TEA.

Fig. 2.

Fig. 2.

A-type currents differed in voltage dependence and kinetics of activation in the four cell types.A, Currents evoked by depolarizing steps (−30 to +30 mV) after a prepulse (1 sec) to −40 mV (left) were subtracted from those evoked by a similar protocol except that a brief (100 msec) prepulse to −95 mV was added immediately before the test steps (middle). Difference currents are shown on the right. Neurons were recorded in the presence of 50 m

m

TEA. B, Activation time constants obtained by fitting the rising phase of the difference currents are shown. Average time constants are plotted as a function of the test pulse voltage for the four cell types. Filled triangles, ChAT/bf neurons (n = 4);open triangles, GP neurons (n = 4); filled circles, ChAT/str neurons (n = 4); and open circles, MS cells (n = 5). C, Plot of the average peak conductance as a function of test pulse voltage for the four cell types is shown. Filled triangles, ChAT/bf neurons (n = 4); open triangles, GP neurons (n = 4);filled circles, ChAT/str neurons (n = 4); and open circles, MS cells (n = 5). Boltzmann fits were obtained for each cell type. D, Application of 0.4 m

m

Cd2+ dramatically reduced the activation rate of A-type current in an MS neuron. Difference currents generated by a step to 0 mV from −95 and −45 mV prepulses are shown.

Fig. 3.

Fig. 3.

A-type currents differed in inactivation voltage dependence and kinetics. A, Conditioning pulses (1.5 sec long ) were used to test the voltage dependence of inactivation.B, Plot of peak current amplitude versus voltage of the conditioning prepulse is shown. Filled triangles, ChAT/bf neurons (n = 6);open triangles, GP neurons (n = 6); filled circles, ChAT/str neurons (n = 6); and open circles, MS cells (n = 5). Thin lines_show Boltzmann fits. C, Semilogarithmic plots of current_traces show that the development of inactivation could be fit by a biexponential function in GP and ChAT/bf neurons and by a monoexponential function in MS and ChAT/str cells. Thin straight lines represent monoexponential fits. Insets, Box plot summaries of rate constants for each cell type are shown: ChAT/bf neurons (n = 6), GP neurons (n = 6), ChAT/str neurons (n = 6), and MS cells (n = 5).

Fig. 4.

Fig. 4.

Recovery from inactivation varied among cell types. The protocol used is depicted at the top of_A_. In ChAT/str neurons, the test pulse was to −20 mV to reduce current amplitude. In all other cell types, the test pulse was to 0 mV. A–C, Examples of recovery of A-type current in a ChAT/str neuron (A), a globus pallidus neuron (B), and a basal forebrain neuron (C) are shown. D, The normalized peak current is plotted against the duration of conditioning prepulse for the examples shown in A–C. In addition, an example from an MS neuron is added. Thin _lines_show biexponential fits.

Fig. 5.

Fig. 5.

Serial dilutions show that Kv4.2 mRNA abundance is correlated with A-type current amplitude. Cell types are arranged from_top_ to bottom in increasing mean A-type current amplitude. Representative single-cell serial dilution gels for Kv4.2 cDNA are shown for each cell type in the left column. Summary distributions for detection thresholds are shown in the column on the right.A, Left, A photo of a gel from a typical MS neuron having a detection threshold of one-quarter (2−2) of the total cDNA is shown. Right, The threshold distribution was best fit with a sum of Gaussian functions (solid line). Inset, A line plot of maximum conductances in a sample of medium spiny neurons is shown. It revealed a high and low conductance group, in accord with the detection data. B, Left, A photo of a gel from a GP neuron in which the detection threshold was one-eighth (2−3) of the total cDNA is shown.Right, The threshold distribution had a mode near this point, but also note the large number of cells in which the transcript was not detected (nd; see open bar).C, Left, A photo of a gel from a typical basal forebrain neuron having a detection threshold of one-sixteenth (2−4) of the total cDNA is shown.Right, The threshold distribution was best fit with a single Gaussian function (solid line) with a mode near the same dilution. D, Left, A photo of a gel from a typical neostriatal cholinergic neuron having a detection threshold of one-sixty fourth (2−6) of the total cDNA is shown. Right, The threshold distribution was best fit with a single Gaussian function (solid line) with a mode near the same dilution. In each gel, the left-hand lane is a sizing ladder. The_right-hand_ lane is for phenotyping cDNAs enkephalin (ENK) and ChAT. The seven lanes in between are PCR products obtained after using an increasing (from left to right) amount of total cellular cDNA (as denoted below) to detect Kv4.2 cDNA.

Fig. 6.

Fig. 6.

Serial dilutions show that Kv4.1 and Kv4.3 mRNA abundance is not correlated with A-type current amplitude. Cell types are arranged from top to bottom in increasing mean A-type current amplitude. Summary distributions for detection thresholds are shown on the left for Kv4.1 and on the right for Kv4.3. A, Summary for MS neurons. The Kv4.1 threshold distribution for MS neurons had a mode near one-quarter of the total single-cell cDNA. The distribution for Kv4.3 detection had a mode nearer one-half of the total cDNA.B, Summary for GP neurons. Note that Kv4.3 was present in high abundance in these neurons. C, Summary for basal forebrain cholinergic neurons. Kv4.1 was the most abundant in this population. D, Summary for neostriatal cholinergic interneurons. Note that Kv4.3 was present at low levels compared with that in GP neurons. nd, Not detected.

Fig. 7.

Fig. 7.

Maximum somatodendritic A-type conductance is directly correlated with estimates of Kv4.2 mRNA abundance.A, Plot of amplicon detection probability as a function of the number of plasmid DNA copies is shown. The solid line represents a fit of Gaussian cumulative function.B, Plot of probability density obtained from the data in_A_ is shown. Each bar represents the normalized change in the probability of amplicon detection corresponding to the change in the mean copy number. C, Average maximal conductance is plotted against the mean estimated number of copies of Kv4.2 cDNA for each of the five cell groups. Copy number estimates were pooled for a group of neurons aspirated without recording as shown in Figure 5. MS neurons were split into high and low abundance groups (see Fig. 5_A_): MS/high (MS/h), n = 12, and MS/low (MS/l), n = 7. Conductance estimates were pooled for a group of neurons in which only phenotyping was done [GP neurons, n = 8; ChAT/bf neurons,n = 9; ChAT/str neurons, n = 6; and MS neurons split into high and low conductance groups (see Fig.5_A_), MS/high, n = 4, and MS/low,n = 5]. The solid line shows the linear regression fit of data points; parameters are shown in the bottom right corner. D, In these experiments, recording and copy number estimates were made from the same cells (GP neurons, n = 6; ChAT/bf neurons,n = 5; ChAT/str neurons, n = 5; and MS neurons, n = 7). As in C, the linear regression is shown as a solid line. Note the similarity with C.

Fig. 8.

Fig. 8.

Kv4.2 cDNA quantification in ChAT neurons using fluorimetric detection. A, Plot of fluorescence as a function of PCR cycle number is shown. Two PCR reactions were performed for each cell. One-fifth of the total cellular cDNA was used for each reaction. Dark lines were generated from ChAT/bf neurons, whereas the lighter lines were generated from ChAT/str neurons. The_horizontal_ line shows the fluorescence threshold. C T is defined as a cycle number at which fluorescence crosses the threshold. B, The solid line is a calibration plot generated with known concentrations of plasmid Kv4.2 cDNA. Superimposed on the line are the mean (± SEM)C T values for ChAT/bf (n = 5) and ChAT/str (n = 4) groups. Arrows indicate the estimated average number of Kv4.2 cDNA copies in one-fifth of the total cellular cDNA in these two cell groups.

References

    1. Akins PT, Surmeier DJ, Kitai ST. Muscarinic modulation of the transient potassium current in rat neostriatal neurons. Nature. 1990;344:240–242. - PubMed
    1. Alonso G, Widmer H. Clustering of KV4.2 potassium channels in postsynaptic membrane of rat supraoptic neurons: an ultrastructural study. Neuroscience. 1997;77:617–621. - PubMed
    1. Audinat E, Lambolez B, Rossier J. Functional and molecular analysis of glutamate-gated channels by patch-clamp and RT-PCR at the single cell level. Neurochem Int. 1996;28:119–136. - PubMed
    1. Baldwin TJ, Tsaur ML, Lopez GA, Jan YN, Jan LY. Characterization of a mammalian cDNA for an inactivating voltage-sensitive K+ channel. Neuron. 1991;7:471–483. - PubMed
    1. Baro DJ, Levini RM, Kim MT, Willms AR, Lanning CC, Rodriguez HE, Harris-Warrick RM. Quantitative single-cell-reverse transcription-PCR demonstrates that A-current magnitude varies as a linear function of shal gene expression in identified stomatogastric neurons. J Neurosci. 1997;17:6597–6610. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources