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. 2002 Jan 1;538(Pt 1):5-23.
doi: 10.1113/jphysiol.2001.013242.

Variable K(+) channel subunit dysfunction in inherited mutations of KCNA1

Ruth Rea  1 , Alexander Spauschus, Louise H Eunson, Michael G Hanna, Dimitri M Kullmann
Affiliations

Variable K(+) channel subunit dysfunction in inherited mutations of KCNA1

Ruth Rea et al. J Physiol. .

Abstract

Mutations of KCNA1, which codes for the K(+) channel subunit hKv1.1, are associated with the human autosomal dominant disease episodic ataxia type 1 (EA1). Five recently described mutations are associated with a broad range of phenotypes: neuromyotonia alone or with seizures, EA1 with seizures, or very drug-resistant EA1. Here we investigated the consequences of each mutation for channel assembly, trafficking, gating and permeation. We related data obtained from co-expression of mutant and wild-type hKv1.1 to the results of expressing mutant-wild-type fusion proteins, and combined electrophysiological recordings in Xenopus oocytes with a pharmacological discrimination of the contribution of mutant and wild-type subunits to channels expressed at the membrane. We also applied confocal laser scanning microscopy to measure the level of expression of either wild-type or mutant subunits tagged with green fluorescent protein (GFP). R417stop truncates most of the C-terminus and is associated with severe drug-resistant EA1. Electrophysiological and pharmacological measurements indicated that the mutation impairs both tetramerisation of R417stop with wild-type subunits, and membrane targeting of heterotetramers. This conclusion was supported by confocal laser scanning imaging of enhanced GFP (EGFP)-tagged hKv1.1 subunits. Co-expression of R417stop with wild-type hKv1.2 subunits yielded similar results to co-expression with wild-type hKv1.1. Mutations associated with typical EA1 (V404I) or with neuromyotonia alone (P244H) significantly affected neither tetramerisation nor trafficking, and only altered channel kinetics. Two other mutations associated with a severe phenotype (T226R, A242P) yielded an intermediate result. The phenotypic variability of KCNA1 mutations is reflected in a wide range of disorders of channel assembly, trafficking and kinetics.

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Figures

Figure 1
Figure 1. Localisation of EA1 mutations and sample traces of two-electrode voltage-clamp measurements
A, schematic representation of a hKv1.1 subunit, showing the positions of the five disease-associated mutations and the TEA tag mutation discussed in this paper (filled circles). Thick outlines mark the positions of the other EA1 mutations identified to date. Each circle represents a single amino acid residue. The dashed rectangle shows the putative transmembrane region of the channel. B–D, sample traces of currents elicited by different voltage protocols in oocytes expressing wt subunits alone, T226R alone, wt and T226R together, or wt*T226R concatemers. B, currents elicited by depolarising voltage steps from −100 mV to +40, 0 or −40 mV. C, time course of deactivation of currents when stepped from +40 mV to 0, −20 or −40 mV. D, currents elicited by 5 s depolarising voltage steps from −100 to 0 mV.
Figure 2
Figure 2. Comparison of whole-cell current properties of fusion proteins (concatemers) with data from studies examining mutant subunits alone or co-expressed with wt subunits
Data for homomeric channels and co-injection experiments are from Spauschus et al. (1999) and Eunson et al. (2000). A, histogram of peak currents (Imax) in response to +40 mV voltage steps from a holding potential of −100 mV. B, threshold of activation as determined by fitting a Boltzmann function to the normalised initial tail current amplitudes, measured at the end of each test pulse when stepping back to −50 mV (V0.5, voltage at half-maximal activation). C, activation time (10–90 % rise time) determined at the beginning of a voltage step to 0 mV. D, characterisation of current deactivation. Following a depolarising pulse to +40 mV, the potential was stepped back to −40 mV. Resulting tail currents were fitted with monoexponentials, and the characteristic time constants plotted. E, degree of C-type inactivation during long depolarising voltage pulses to 0 mV. The amplitude of the current at the end of the 5 s pulse was normalised to the initial peak current amplitude. Note the logarithmic ordinate scale in C and D. All oocytes were injected with cRNAs as indicated. The horizontal grey bands indicate the parameter values (±1 s.e.m.) obtained for wt and wt*wt concatemers. All error bars indicate s.e.m. (n ranged from 5 to 29).
Figure 3
Figure 3. TEA sensitivity reveals the relative contribution of wt and TEA-tagged subunits to whole-cell currents
A, TEA sensitivity of homomeric wt and wtT channel currents (see insets). Data points for wt, wtT and wt*wtT (n = 23, 28 and 13, respectively) are fitted by eqn (7), with apparent equilibrium dissociation constants Ki,0 = 0.4 mm, Ki,4 = 250 mm and Ki,2 = 10 mm, respectively. Dose-response data obtained by co-expressing wt + wtT (n = 12) are fitted by eqn (9) (where f = 0.5), which describes a binomial distribution of the five possible channel stoichiometries, with equal probability of incorporation of wt and wtT subunits. Equation (10), which describes the dose-response curve expected if wt and wtT subunits formed two separate populations of homomeric channels, fails to fit the data. B-F, dose-response data obtained by co-expressing each TEA-tagged mutant (mutantT) with wt subunits. For wt + T226RT (n = 17; B), A242PT (n = 7; C) and R417stopT (n = 14; F), the points are shifted to the left of the curve given by eqn (9) (with f = 0.5), implying that mutantT subunits contribute less to the whole-cell current than wt subunits. In all cases, eqn (10) (representing two separate populations of homomeric channels), fails to fit the co-expression data, implying that mutant subunits co-assemble with wt. Error bars indicate s.e.m.
Figure 4
Figure 4. R417stop affects both channel assembly and tetramer function
Aa, Ba, Ca, TEA sensitivity obtained by co-expressing either wt and R417stopT subunits (n = 14), or wtT and R417stop subunits (n = 12). Ab, Bb, Cb, maximal current amplitudes for wt and R417stopT. TEA dose-response data obtained by expressing wt*R417stopT concatemers (n = 14) are also shown in Aa, but are omitted from Ba and Ca for clarity. The curves used to fit the dose-response of homomeric wt, wtT, wt*wtT and wt + wtT currents (see Fig. 3) are also shown for reference. A, the TEA sensitivity can be fitted by increasing f in eqn (11) to 0.77, representing impaired assembly of R417stopT subunits. However, because the function of assembled tetramers is assumed to be unaffected by the presence of mutant subunits (i0 = i1 =… i4 = 1), the current amplitude data are not fitted. B, the current carried by each tetramer is assumed to vary according to its stoichiometry (im optimised), but tetramerisation is assumed to be unaffected by the mutation (f = 0.5). Although this model agrees with the amplitude data, the TEA sensitivity cannot be fitted. C, the TEA sensitivity and amplitudes are fitted simultaneously by allowing both the efficacy of assembly and subsequent stages of expression to vary (f and im both optimised). All error bars indicate s.e.m.
Figure 5
Figure 5. T226R, V404I and R417stop affect channel open probability at 0 mV
A, examples of current responses to 1 s voltage steps from −80 to 0 mV. Single channels were composed of mutant and wt subunits in a fixed 1:1 ratio (concatemers). Dotted lines indicate the closed state of the channel. B, histogram of the slope conductance measured at 0 mV. C, histogram of the channel open probability at 0 mV. ** P < 0.05 (tested against wt).
Figure 6
Figure 6. EGFP tagging of wt and R417stop to examine protein expression
Aa, example traces of EGFP-Kv1.1 currents elicited by depolarising steps between −60 and +40 mV, from a holding potential of −100 mV. Ab, example trace of an EGFP-R417stop current elicited by stepping from −100 to +40 mV. B, example images of the membrane (m) or cytoplasmic (c) side of halved oocytes injected with EGFP-Kv1.1 (a and b), EGFP-R417stop (c and d) or water (e and f), obtained using fluorescence microscopy. Dotted lines indicate the region of fluorescence measurement. Scale bar, 250 μm. C, bar charts showing the mean fluorescence pixel value (F) obtained from oocytes imaged on the membrane side (a), cytoplasmic side (b), or averaged across the whole cell (c), normalised to the mean fluorescence signal obtained for EGFP-Kv1.1 (FEGFP-Kv1.1). Oocytes were injected with 1 unit of EGFP-Kv1.1 cRNA (n = 10), EGFP-R417stop cRNA (n = 4), EGFP-Kv1.1 cRNA together with 1 unit of non-fluorescent R417stop cRNA (EGFP-Kv1.1 + R417stop; n = 5), EGFP-R417stop cRNA together with non-fluorescent wt cRNA (EGFP-R417stop + wt; n = 5), or H2O (n = 3). Asterisks denote a significant difference of P < 0.05 between the conditions indicated by the dotted lines (Student's t test). All error bars indicate s.e.m.
Figure 7
Figure 7. Co-expression of R417stop with wild-type Kv1.2 subunits
A, example traces of currents elicited by depolarising voltage steps between −60 and +40 mV, from a holding potential of −100 mV, in a cell injected with Kv1.2 alone (a) or with Kv1.2 plus R417stop in a 1:1 unit ratio (b). B, mean peak current amplitudes in cells expressing Kv1.2 alone, or Kv1.2 co-expressed with R417stop in a 1:1, 1:4 or 1:8 ratio. The same number of Kv1.2 cRNA copies was injected in each condition, except where ‘2 units’ is indicated, where the number of cRNA copies was doubled to show that current saturation levels were not reached. C, TEA dose-response data of currents produced by expression of Kv1.2 alone (n = 4), Kv1.2 with R417stop in a 1:1 ratio (n = 5), or Kv1.2 with Kv1.1 in a 1:1 ratio (n = 5). Data points show the mean dose-response in each condition. Curves are taken from Fig. 4 and are included for comparison of Kv1.2 data with the results of Kv1.1 modelling. All error bars indicate s.e.m.
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