©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Modulation of Cardiac Na Channel Expression in Xenopus Oocytes by 1 Subunits (*)

(Received for publication, June 9, 1995)

Yusheng Qu Lori L. Isom (§) Ruth E. Westenbroek John C. Rogers (¶) Timothy N. Tanada Kimberly A. McCormick Todd Scheuer William A. Catterall

From the Department of Pharmacology, Box 357280, University of Washington, Seattle, Washington 98195-7280

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Voltage-gated Na channels consist of a large alpha subunit of 260 kDa associated with beta1 and/or beta2 subunits of 36 and 33 kDa, respectively. alpha subunits of rat cardiac Na channels (rH1) are functional when expressed alone in Xenopus oocytes or mammalian cells. beta1 subunits are present in the heart, and localization of beta1 subunit mRNA by in situ hybridization shows expression in the perinuclear cytoplasm of cardiac myocytes. Coexpression of beta1 subunits with rH1 alpha subunits in Xenopus oocytes increases Na currents up to 6-fold in a concentration-dependent manner. However, no effects of beta1 subunit coexpression on the kinetics or voltage dependence of the rH1 Na current were detected. Increased expression of Na currents is not observed when an equivalent mRNA encoding a nonfunctional mutant beta1 subunit is coexpressed. Our results show that beta1 subunits are expressed in cardiac muscle cells and that they interact with alpha subunits to increase the expression of cardiac Na channels in Xenopus oocytes, suggesting that beta1 subunits are important determinants of the level of excitability of cardiac myocytes in vivo.


INTRODUCTION

Cardiac Na channels are responsible for the rapid, depolarizing upstroke in the cardiac action potential. Their function is critical for the rapid spread of depolarization through the heart and, ultimately, for cardiac contractility. Cardiac Na channels have properties that distinguish them from other well characterized voltage-dependent Na channels. They are less sensitive to tetrodotoxin (Baer et al., 1976), and their kinetics of activation and inactivation are slower and more complex (Brown et al., 1981). A Na channel alpha subunit cDNA encoding this channel has been isolated from newborn rat heart (rH1) (^1)(Rogart et al., 1989) and denervated rat skeletal muscle (Skm2, Kallen et al.(1990)) and a closely related Na channel has been isolated from human cardiac muscle (Gellens et al., 1992). Expression of these alpha subunit cDNAs in Xenopus oocytes (Cribbs et al., 1990; White et al., 1991; Gellens et al., 1992) and mammalian cells (Qu et al., 1994; O'Leary and Horn, 1994) yields Na currents with functional properties and tetradotoxin sensitivity characteristic of the native cardiac Na channel.

In electric eel electroplax, Na channels are composed of a single large alpha subunit with a molecular mass of 230-270 kDa (Agnew et al., 1980; Miller et al., 1983; Norman et al., 1983). The major form of the Na channel in rat brain is a heterotrimeric complex of an alpha subunit (260 kDa), a noncovalently bound beta1 subunit (36 kDa), and a disulfide-linked beta2 subunit (33 kDa) (Catterall, 1992). Na channels in rat skeletal muscle are heterodimeric, composed of an alpha subunit and only one beta subunit (Barchi, 1983; Kraner et al., 1985) which is encoded by the same gene as the brain beta1 subunit (Makita et al., 1994). Currents due to brain or skeletal muscle Na channel alpha subunits expressed alone by injection of mRNA in Xenopus oocytes are small and have abnormally slow kinetics (Auld et al., 1988; Krafte et al., 1988; Joho et al., 1990; Krafte et al., 1990). Coexpression of the beta1 subunit increases channel expression, shifts the gating mode from slow to fast, speeds activation and inactivation kinetics, and causes a hyperpolarizing shift in the voltage dependence of activation and inactivation (Isom et al., 1992; Cannon et al., 1993; Makita et al., 1994; Patton et al., 1994; Isom et al., 1995). Thus, beta1 subunits both modify the functional properties of brain and skeletal muscle Na channels and increase the efficiency of their expression.

The role of the beta1 subunit in the heart has been less clear. Initial studies using subunit-specific antibodies identified the beta1 subunit polypeptide in the heart (Sutkowski and Catterall, 1990). beta1 subunit mRNA also has been identified in heart (Isom et al., 1992; Tong et al., 1993; Yang et al., 1993; Makita et al., 1994) and cDNAs homologous to the rat brain beta1 transcript have been cloned from rat cardiac muscle (Bennett et al., 1993; McClatchey et al., 1993; Yang et al., 1993). The human and rat cardiac beta1 cDNAs are identical in sequence to their brain counterparts (Makita et al., 1994). (^2)However, purified preparations of cardiac Na channels from chicken and rat do not have associated beta1 subunits (Lombet and Lazdunski, 1984; Cohen and Levitt, 1993), and there have been conflicting reports concerning the functional significance of beta1 subunits in the heart. Yang et al.(1993) reported no effect of beta1 expression, while Kyle et al.(1993) observed a depolarizing shift in the voltage dependence of inactivation as a result of beta1 coexpression.

In these experiments, we have investigated whether beta1 subunit mRNA is expressed in cardiac muscle cells using in situ hybridization, and we have examined the functional role of beta1 subunits in modulating cardiac Na channel expression and kinetics by coexpression of mRNA for alpha and beta1 subunits in Xenopus oocytes. We report that beta1 subunit mRNA is expressed in cardiac muscle cells in vivo. When expressed in Xenopus oocytes in conjunction with the rH1 alpha subunit, beta1 subunits substantially increase Na channel expression, but have no detectable effects on physiological properties. Thus, beta1 subunits are likely to be important determinants of the level of functional expression of Na channels in cardiac cells, but do not alter their physiological properties significantly.


EXPERIMENTAL PROCEDURES

Construction of Expression Vectors

A full-length cDNA encoding the rH1 Na channel alpha subunit was assembled in pBluescript SK by polymerase chain reaction amplification and sequencing of cDNA fragments using primers designed from the rH1 sequence (Rogart et al., 1989; Qu et al., 1994). (^3)The full-length alpha subunit cDNA was then cloned into the BglII site of pSP64T. The oocyte expression vector pbeta1.SP64T was constructed as described previously (Patton et al., 1994). Both vectors contained only the coding sequence of the cDNAs.

The deletion mutant beta1 DeltaVal138-Ser159 was constructed by first removing a portion of the 5`-untranslated region of pbeta1.C1Aa (Isom et al., 1992) by deletion of nucleotides 1 through 175. This region is predicted to contain stem-loop structures which may decrease the efficiency of beta1 expression (Patton et al., 1994). Single stranded DNA was prepared by interference helper phage VCS-M13 infection of XL1-blue transformants (Stratagene, La Jolla, CA), and served as template for oligonucleotide-directed ``loop out'' deletion mutagenesis. A 36-base ``clamp'' oligonucleotide was designed to anneal to two segments of the sense strand template which flanked the region to be deleted. In vitro mutagenesis reactions were performed as described for the Sculptor IVM system (Amersham Corp.). The resulting mutant phagemid contained a 66-base pair deletion of the nucleotides encoding Val through Ser. The deleted amino acids are located in the extracellular domain of the beta1 polypeptide, adjacent to the proposed transmembrane segment, and do not contain a putative glycosylation site.

mRNA Transcription

pSP64T.rH1.2 was linearized with NotI and pbeta1.C1Aa was linearized with EcoRI and transcription was performed using the Ambion SP6 mMessage mMachine kit according to the manufacturer's instructions. The quality of the RNA from each transcription reaction was checked by agarose gel electrophoresis, and the RNA dissolved in nuclease-free water for oocyte injection.

Expression of Constructs and Electrophysiological Recording

Ovarian lobes were removed from adult female Xenopus laevis and the follicular layers were digested using collagenase (2.3 mg/ml, Sigma, Type I) dissolved in OR-2 (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl(2), 5 mM HEPES, pH 7.5). After digestion, healthy oocytes were manually selected based upon size and uniformity of color and incubated at 18 °C in Barth's medium (88 mM NaCl, 1 mM KCl, 0.82 mM MgSO(4), 0.33 mM Ca(NO(3))(2), 0.41 mM CaCl(2), 2.4 mM NaHCO(3), 10 mM HEPES, pH 7.4, 50 µg/ml gentamicin, and 5% fetal bovine serum). Twenty-four h after collagenase digestion, oocytes were again selected and injected with the mRNA mentioned above. The oocytes were studied 3 days after injection.

For two-microelectrode voltage-clamp experiments, the oocytes were continuously perfused at room temperature (23-25 °C) with Ringer's solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl(2), 10 mM HEPES, pH 7.2). Whole cell Na currents were studied using the conventional two-microelectrode voltage-clamp technique (Patton and Goldin, 1991) with a CA-1 amplifier (DAGAN). For cell-attached macropatch recordings, oocytes were first manually stripped with fine forceps under a dissecting microscope after shrinking with a hypertonic solution (200 mM potassium aspartate, 20 mM KCl, 10 mM EGTA, 1 mM MgCl(2), 20 mM HEPES, pH 7.4-7.5; Methfessel et al.(1986)). During recordings, the oocytes were bathed in a high K solution (110 mM KCl, 10 mM NaCl, 10 mM EGTA, 1 mM MgCl(2), 10 mM HEPES, pH 7.2) to bring the membrane potential to approximately 0 mV. The electrode tip was coated with Sylgard (Dow Corning) and filled with 150 mM NaCl, 1.5 mM CaCl(2), 2 mM MgCl(2), 5 mM HEPES, pH 7.4 (tip resistance 0.5-1 m). Macropatch current was recorded in the cell-attached configuration (Hamill et al., 1981) using an AxoPatch-1C amplifier (Axon Instruments). The voltage-clamp protocols are described in figure legends or corresponding text. Conductance-voltage (g-V) relationships were calculated from current-voltage (I-V) relationships according to g = I/(V-V(r)), where I is the peak current measured at voltage V and V(r) is the measured reversal potential. Normalized conductance-voltage relationships and inactivation curves were fit with a Boltzmann distribution, 1/(1 + exp[(V-V)/k]), where V is the voltage of half-activation or half-inactivation and k is a slope factor. Pooled data are reported as means ± S.E. Statistical comparisons were made using Student's t test, with p < 0.05 taken as the critierion of significance.

In Situ Hybridization Analysis of Rat Cardiac beta1 Subunit mRNA

A probe for in situ hybridization analysis of cardiac beta1 subunit mRNA expression was generated by polymerase chain reaction using the following conditions: 1 ng of human genomic DNA template, 4 mM each primer (forward: GGGCTGCGTGGAGGTGG, reverse: TCTTGTGCAGCAGCTTC), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgSO(4), 0.001% (w/v) gelatin, 200 mM each deoxyribonucleotide triphosphate, and 2.5 units of Taq DNA polymerase (Cetus). The amplification was performed as follows: 1 min at 90 °C; 1 min 15 s at 50 °C; 2 min at 72 °C for 40 cycles followed by 10 min at 72 °C and stopping of the reaction at 4 °C. The resulting 527-nucleotide polymerase chain reaction product was sequenced using Sequenase (U. S. Biochemical Corp.), subcloned into pBluescript SK, and linearized with the appropriate restriction endonucleases to generate sense and antisense probe templates, respectively. This sequence was identical to that reported previously for rat brain beta1 (Isom et al., 1992) except for the following changes: A for G at residue 312, T for A at residue 484, T for C at residue 629, and T for G at residue 753. Transcription reactions were carried out with either T3 or T7 RNA polymerase incorporating digoxigenin-11-UTP and quantitated according to the Genius System product literature (Boehringer Mannheim).

In situ hybridization of free-floating sections was carried out using modifications of methods described previously (Miller et al., 1989; Black et al., 1994). Briefly, adult Sprague-Dawley rats were anesthetized using sodium pentabarbitol, the heart was removed, immediately frozen in powdered dry ice, and stored at -70 °C. Sagittal sections (40 µm) through the long axis of the heart were cut on a sliding microtome and then placed into 4% paraformaldehyde fixative for 45 min. Tissue sections were then rinsed in 0.05 M phosphate-buffered saline for 20 min, water for 2 min, 0.1 M triethanolamine, pH 8.0, for 2 min and then in 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min to reduce nonspecific binding. The tissue sections were then rinsed in 2 times SSC for 10 min, 70% ethanol for 2 min, 95% ethanol for 2 min, 100% ethanol for 2 min, 70% ethanol for 2 min, and finally in water for 2 min. Sections were prehybridized for 3 h at 42 °C in a buffer containing 44.6% formamide, 8.9% dextran sulfate, 0.27 M NaCl, 7 mM Tris, pH 8.0, 0.7 mM EDTA, 0.9 times Denhardt's, 16 mM dithiothreitol, 0.45 mg/ml yeast tRNA, and 4.6 times Genius Northern blocking solution (Boehringer Mannheim). The tissue was then transferred to hybridization solution containing 41% formamide, 8.2% dextran sulfate, 0.25 M NaCl, 6.5 mM Tris, pH 8.0, 0.66 mM EDTA. 0.82 times Denhardts, 14.8 mM dithiothreitol, 1.2 µg/ml yeast tRNA, 4.1 times Genius Northern blocking solution, and 7.5 µg/ml of the digoxigenin-labeled probe and incubated overnight at 42 °C. Tissue sections were rinsed in 1 times standard sodium chloride/sodium citrate (SSC; Miller et al. 1989) for 30 min, treated with 25 µg/ml RNase A in RNase buffer (10 mM Tris, 0.5 M NaCl, and 1 mM EDTA) for 30 min, rinsed in 1 times SSC for 30 min, rinsed in 0.1 times SSC at 45 °C for 40 min, rinsed in 0.1 times SSC at room temperature for 15 min, and then in 0.1 M Tris-buffered saline, pH 7.4, for 30 min. The tissue was then blocked using 10% normal sheep serum in 0.1 M Tris-buffered saline for 1 h at room temperature before being placed in alkaline phosphatase-conjugated anti-digoxigenin F(ab) antibody (diluted 1:400 in 0.1 M Tris-buffered saline containing 10% normal sheep serum and 0.1% Triton X-100) for 24 h at room temperature. The tissue was then washed in 0.1 M Tris-buffered saline for 1 h followed by 1 h of rinsing in a solution containing 100 mM Tris, 50 mM MgCl(2), and 100 mM NaCl, pH 9.5. The tissue was then reacted with 4-nitro blue tetrazolium chloride (0.45 mg/ml), 5-bromo-4-chloro-3-indolyl phosphate (0.175 mg/ml), and levamisole (0.24 mg/ml) in the reaction buffer, rinsed in stop buffer (10 mM Tris and 1 mM EDTA, pH 8.0) for 30 min and the free floating sections were finally mounted on gelatin-subbed slides, air dried, coverslipped using Biomeda gel mount (Fischer), and viewed using a Leitz Dialux microscope.

Control experiments included omitting probes from hybridization solution and substituting sense probes for antisense probes. In addition, beta1 antisense probe was hybridized to liver tissue to test for nonspecific labeling with the probe. No specific labeling was observed in the controls.


RESULTS

In Situ Hybridization of beta1 in Heart

beta1 subunit mRNA and protein have been detected in the heart using immunoblotting and Northern blotting methods (Sutkowski and Catterall, 1990; Isom et al., 1992; Tong et al., 1993; Yang et al., 1993; Makita et al., 1994), but it is not known whether this beta1 mRNA is in the neurons resident in the heart or in the cardiac myocytes themselves. The cellular distribution of sodium channel beta1 subunit mRNA was examined in heart using non-isotopic in situ hybridization methods. At high magnification there is positive staining of muscle cells in regions of the cytoplasm surrounding the nucleus (Fig. 1, A-C). No staining was observed when the sense probe was used or when no probe was added to the hybridization solution (Fig. 1D). As a further control, liver tissue was also stained with the beta1 probe and, as expected, there was no staining. Lower magnification views show that beta1 is expressed in myocytes throughout the heart with the most prominent staining located in the papillary muscles of the ventricles. These results demonstrate the presence of beta1 mRNA within most cardiac muscle cells.


Figure 1: In situ hybridization of beta1 mRNA in heart. Adult rat heart sections were processed for in situ hybridization as described under ``Experimental Procedures.'' A, ventricular papillary heart tissue hybridized with beta1 antisense probe demonstrating the presence of beta1 mRNA in muscle cells. Arrowheads outline the nuclei. B and C, higher magnifications of heart tissue hybridized with beta1 antisense probe illustrating that labeling is present in the cytoplasm surrounding the nucleus. D, tissue section hybridized with beta1 sense probe to illustrate the lack of hybridization and the specificity of the beta1 antisense probe. Scale bars: A equals 20 µm; B-D equal 10 µm.



Coinjection of Rat Brain beta1 Subunit mRNA Increased Cardiac NaChannel Expression in Xenopus Oocytes

To investigate the functional consequences of coexpression of beta1 subunits with cardiac rH1 alpha subunits, we injected mRNAs encoding the two subunits into Xenopus oocytes. Na currents in the oocytes were measured 72 h after mRNA injection using two-microelectrode voltage clamp. At a single concentration of alpha subunit, Na currents were dramatically larger at all test potentials in oocytes coinjected with beta1 subunit mRNA (Fig. 2).


Figure 2: Effect of rat brain Na channel beta1 subunit mRNA co-injection with rH1 alpha subunit mRNA on expressed Na currents in Xenopus oocytes. Currents were recorded in oocytes injected with alpha alone (25 ng/µl, left) or alpha (25 ng/µl) + beta1 (50 ng/µl, right) mRNA during pulses from a holding potential of -100 mV to potentials ranging from -45 to 0 mV in 5-mV increments using two-microelectrode voltage clamp.



To examine the concentration dependence of this increase in Na current amplitude, beta1 RNA was injected at concentrations ranging from 10 to 500 ng/µl with a constant 25 ng/µl of alpha subunit RNA. The amplitude of the expressed Na current increased as the amount of beta1 RNA injected was increased (Fig. 3A). No current was observed when beta1 subunits were injected in the absence of the alpha subunit. These results were obtained in one series of oocytes injected simultaneously and studied at times ranging from 62 to 78 h after injection. Similar results were obtained in two other experimental series of this kind.


Figure 3: Effect of beta1 subunit mRNA coinjection on rH1 Na current expression. Mean current levels were measured 72 h after injection in a single batch of oocytes (n = 6 for each point). A, effects of beta1 mRNA concentration. Each point was significantly different from the adjacent one (p < 0.05) except for the last two right-hand bars with the highest beta1 mRNA concentrations. B, comparison of effects of wild type and mutant beta1 subunit on Na channel expression. rH1 alpha mRNAs was injected into oocytes with or without beta1 mRNA or with an inactive mutant beta1 mRNA (DeltaVal-Ser) with those amino acids deleted. The amounts of alpha and beta1 mRNAs injected are indicated beneath the histogram bars.



Significant increases in Na channel current amplitude were observed at a beta1:alpha RNA molar ratio of 5:3. The observed increase in current amplitude saturated at a beta1:alpha RNA molar ratio of 50:1, with a half-maximal increase at a beta1:alpha molar ratio of 10:1 (equivalent to a weight ratio of 2:1). The maximal increase in current induced by beta1 subunit RNA coinjection was 3-6-fold, depending on the batch of oocytes (mean = 5.2 ± 1.4-fold, n = 22 oocytes). This is comparable to the increase observed when rat brain type IIA Na channel alpha subunits were coinjected with beta1 subunit mRNA in Xenopus oocytes (Isom et al., 1992) and to the increase in [^3H]saxitoxin binding observed as a result of type IIA alpha subunit and beta1 subunit coexpression in transfected mammalian cells (Isom et al., 1994).

An alternative explanation for the observed increase in Na current amplitude is that beta1 subunit mRNA may exert nonspecific effects on Na channel expression or mRNA expression in general. To examine that possibility, a deletion mutant of rat brain beta1 (beta1DeltaVal-Ser) was constructed as described under ``Experimental Procedures.'' The functional effects of this mutant beta1 subunit were tested by coinjecting it with the rat brain type IIA alpha subunit (beta1:alpha molar ratio of 100:1). The macroscopic Na current time courses from oocytes injected with transcripts encoding the rat brain type IIA alpha subunit alone and in conjunction with beta1DeltaVal-Ser were very similar. Current-voltage relationships, steady-state inactivation curves, and the time course of recovery from inactivation also were unaffected (data not shown). This indicates that beta1DeltaVal-Ser is inactive, probably because it does not associate with the alpha subunit. Coinjection of beta1DeltaVal-Ser with rH1 alpha subunit mRNA (10:1 molar ratio) caused no increase in current amplitude (Fig. 3B). Wild-type beta1 subunits caused the normal increase in current amplitude in the same batch of oocytes (Fig. 3B). Similar results were observed in two other experiments. Thus, an active beta1 subunit is necessary to observe the increase in current with beta1 subunit RNA coinjection.

Coinjection of beta1 Subunit RNA Did Not Change the Electrophysiological Properties of rH1 NaCurrent

Electrophysiological effects of the beta1 subunit were examined by recording currents in Xenopus oocytes due to injection of the rH1 alpha subunit RNA alone or in combination with beta1 subunit mRNA. To insure adequate time and voltage control, recordings were made of macroscopic currents in small membrane patches (macropatches) that allow excellent time and spatial control of membrane potential. The effect of beta1 subunits on expression of functional Na channels was verified by contemporaneous two-microelectrode voltage clamp recordings from oocytes in the same batch used for kinetic comparisons. An average increase in current amplitude of 3-4-fold was observed in these beta1-coinjected oocytes. Fig. 4A shows macropatch recordings from oocytes injected with alpha subunit RNA alone (500 ng, left panel) or in conjunction with beta1 subunit RNA (100 ng of alpha, 200 ng of beta1, right panel). Little overall difference in Na current kinetics was observed. To detect quantitative effects on current time course, normalized and averaged currents measured during test pulses to -60, -40, -20, or +20 mV were compared (Fig. 4B). Average time courses obtained with (dotted line) and without (solid line) coinjection of beta1 subunit mRNA were virtually superimposable. Inactivation time courses at +20 mV with and without beta1 were also compared by fitting the decaying phase of the current with 2 exponentials. In oocytes injected with alpha subunit RNA alone, the time constant of the fast component (1) was 0.42 ± 0.12 ms with an amplitude of 93% and the time constant of the slow component (2) was 3.4 ± 1.6 ms (n = 7). In oocytes coinjected with beta1 subunit mRNA, 1 was 0.48 ± 0.08 ms with an amplitude of 94% and 2 was 2.9 ± 1.5 ms (n = 8). An unpaired Student's t test showed no significant difference with all three parameters (p > 0.05). Despite having large effects on Na current amplitude, coinjection of beta1 subunits had little effect on current time course.


Figure 4: Effects of coinjection of beta1 subunit mRNA on the time course of rH1 Na current. A, current traces recorded in cell-attached macropatch configuration from oocytes injected with alpha subunit mRNA alone (500 ng/µl, left) and in oocytes coinjected with beta1 subunit mRNA (alpha 100 ng/µl + beta1 200 ng/µl, right) during depolarizations to -80, -70, -60, -50, -40, -30, -20, -10, 0, 20, 60, and 80 mV from a holding potential of -120 mV. B, averages of macropatch current traces during pulses to the indicated voltages from oocytes injected with rH1 alpha subunit mRNA alone (solid lines, n = 18) or coinjected with beta1 subunit mRNA (dotted lines, n = 15). The average current traces shown were constructed by normalizing the amplitudes of current traces from individual experiments and then averaging them.



Effects of beta1 subunits on the voltage dependence of Na channel activation and inactivation were determined using both two-microelectrode and cell-attached patch recording configurations. Mean voltage dependence of Na current activation was unaffected by coexpression of the beta1 subunit with the alpha subunit in cell-attached patches or in two-microelectrode recordings (Fig. 5, A and B). The voltage dependence of Na channel inactivation was determined using conditioning prepulses (98 ms long in cell-attached experiments, 500 ms long in two-microelectrode experiments) followed by a test depolarization. The mean voltage dependences of Na channel activation and inactivation were not significantly affected in either recording configuration due to coinjection of beta1 subunit RNA (Fig. 5, A and B). The voltage dependences of activation and inactivation determined in the cell-attached configuration with or without beta1 subunits were shifted negatively compared to two-microelectrode recordings as has been previously observed for the native cardiac channel (Kimitsuki et al., 1990).


Figure 5: Effects of beta1 subunit coinjection on the voltage dependence of rH1 Na current activation and inactivation and on its time course of recovery from inactivation. Determinations were either from cell-attached macropatches (A and C) or from two-microelectrode recordings (B and D). A and B, mean voltage dependences of activation and inactivation with () and without (bullet) coinjection of beta1 subunits. The data plotted correspond to mean values derived from Boltzmann fits to individual experiments. In the cell-attached configuration the mean values for alpha alone (bullet) were: Vact = -50.4 ± 5.9 mV with slope factor, k = -7.4 ± 1.0 mV (n = 18), Vinact = -99.8 ± 9.4 mV, k = 9.1 ± 4.0 (n = 17); alpha + beta1 (): Vact = -53.0 ± 6.2 mV, k = -7.3 ± 0.6 (n = 15), Vinact = -97.3 ± 9.4 mV, k = 9.4 ± 1.8 (n = 13). For two-microelectrode recordings, alpha alone (bullet): Vact = -27.8 ± 2.3 mV, k = -6.2 ± 0.6 mV (n = 12), Vinact = -52.0 ± 4.2 mV, k = 6.5 ± 1.0 (n = 6); alpha + beta1 (): Vact = -28.7 ± 1.6 mV, k = -6.0 ± 0.8 (n = 10), Vinact = -56.1 ± 3.5 mV, k = 6.5 ± 1.0 (n = 14). C and D, recovery from inactivation in oocytes injected with rH1 alpha subunit mRNA alone (bullet) and in conjunction with beta1 subunit mRNA (). C, recovery at -120 mV following a 16-ms pulse to -30 mV. Normalized peak currents from each data set were fit with single exponentials, alpha alone: = 7.81 ms; alpha + beta(1): = 6.19 ms. D, recovery at -100 mV after a 5-s conditioning pulse to -10 mV. Both data sets were fitted with two exponentials, alpha alone: (1) = 6.23 ms, (2) = 1120.06 ms, A(1) = 0.45, A(2) = 0.55; alpha + beta(1): (1) = 6.50 ms, (2) = 854.28 ms, A(1) = 0.44, A(2) = 0.56.



Recovery from inactivation was also studied in both the cell-attached and two-microelectrode recording configurations using double-pulse protocols. In the cell-attached configuration, Na channels were inactivated using a 16-ms conditioning pulse to -30 mV. The membrane potential was then returned to -120 mV to allow channels to recover from inactivation. The degree of recovery was assessed at various recovery times with a test pulse to -30 mV. Recovery time courses at -120 mV determined by plotting normalized peak test pulse current versus recovery time were well fit with single exponentials (Fig. 5C). In oocytes injected with alpha subunit RNA alone, = 6.9 ± 1.8 ms, and in oocytes coinjected with beta1 subunit mRNA, = 7.3 ± 1.4 ms (n = 4). For recovery experiments in the two-microelectrode configuration, the conditioning pulse was 5 s long and recovery was studied at -100 mV. Such a long prepulse is expected to generate both fast and slow inactivation of the Na channel. After such prepulses, fits of the recovery time course required two exponential components (Fig. 5D). In oocytes injected with alpha alone, the time constant of the fast component (1) was 5.5 ± 3.1 ms, and the time constant of the slow component (2) was 1010 ± 174 ms, with initial amplitudes, A1 and A2, of 52 and 48% respectively (n = 3). In oocytes coinjected with beta1 subunit mRNA, 1 was 6.0 ± 0.9, 2 was 868 ± 163, with A1 and A2 each equaling 50% (n = 3). Differences between results with alpha alone and with beta1 coinjection were insignificant (unpaired Student's t test, p > 0.05). Consistent with the lack of effect on recovery from inactivation, after a 10 Hz train of 20 15-ms long pulses to -10 mV from a holding potential of -100 mV, current was reduced to 92.4 ± 1.8% of its initial value with rH1 alpha alone and to 91.9 ± 1.9% with coinjection of beta1 (n = 7). Again, these values were not significantly different from each other (p > 0.05). Thus, in oocytes where effects of beta1 subunit RNA coinjection have been verified by recordings of significantly increased Na current levels, there was no significant difference in Na current time course, voltage dependence of activation, voltage dependence of inactivation, or recovery from inactivation when comparing Na currents due to injection of rH1 Na channel alpha subunit RNA alone or when coinjected with beta1 subunit RNA.


DISCUSSION

Our results show that beta1 subunit mRNA is expressed in cardiac muscle cells as assessed by high resolution in situ hybridization. Thus, beta1 subunits are available to modulate rH1 Na channels in vivo. These studies complement previous work showing that beta1 subunit mRNA and protein are present in the heart without defining the cell-type expressing them (Sutkowski and Catterall, 1990; Isom et al., 1992; Tong et al., 1993; Yang et al., 1993; Makita et al., 1994).

Coexpression of rH1 alpha subunits and beta1 subunits in Xenopus oocytes substantially increases the level of Na currents. Since both beta1 subunits and alpha subunits are present in cardiac myocytes, it is likely that beta1 subunits associate with alpha subunits and increase the expression of functional Na channels in cardiac cells as well. Purified Na channels from chicken and rat heart do not have associated beta1 subunits (Lombet and Lazdunski, 1984; Cohen and Levitt, 1993). However, beta1 subunits dissociate easily from detergent-solubilized Na channels (Messner and Catterall, 1986) and therefore may have been lost in purification. Since the purified preparations of Na channels from heart have not been functionally reconstituted, it remains to be determined whether these preparations which appear to lack beta1 subunits can function in voltage-activated ion conductance.

Little effect of beta1 subunits was observed on the kinetics and voltage dependence of expressed current. The lack of kinetic effects was consistent with effects of human heart beta1 subunits on human heart Na channel alpha subunit (Makita et al., 1994). It differs from reports that coinjection of the rat brain beta1 subunit RNA with the rat heart Na channel alpha subunit RNA causes a 3 or 6 mV depolarizing shift in the voltage dependence of inactivation (Kyle et al., 1993; Makielski et al., 1995) and from a report that beta1 subunits cause a 3-mV hyperpolarizing shift in the voltage dependence of inactivation (Nuss et al., 1995). These small differences in results may reflect differences in the details of the experimental procedures used in the different studies.

Injection of cardiac (rH1) alpha subunit mRNA into oocytes gave currents with comparable kinetics and voltage dependence to Na currents in native cardiac myocytes (Satin et al., 1992). Recordings from our laboratory in neonatal rat ventricular myocytes (Qu et al., 1994) gave half-activation values of -34 mV and half-inactivation values of -58 mV. Currents expressed in oocytes after injection of rH1 alpha subunit RNA were half-activated at -28 mV and half-inactivated at -52 mV (see Fig. 5B), only 6 mV more positive than Na currents in ventricular myocytes. These small differences contrast sharply with injection of brain or muscle Na channel alpha subunit RNA alone into Xenopus oocytes. Those currents exhibit abnormally slow kinetics and positively shifted voltage dependences of activation and inactivation which were normalized by coinjection of beta1 subunits (Isom et al., 1992; Makita et al., 1994). Thus, beta1 subunits have much more striking functional effects on brain and skeletal muscle Na channels than on cardiac Na channels.

During development of both rat retinal ganglion cells and rat forebrain neurons in vivo, expression of beta subunits and their assembly with alpha subunits is concurrent with a 5- to 10-fold increase in the number of Na channels (Wollner et al., 1988; Scheinman et al., 1989; Sutkowski and Catterall, 1990). Thus, expression and assembly of beta subunits may be a rate-limiting step in Na channel expression. In addition, beta1 subunits stabilize purified and reconstituted brain Na channels. Channel function was completely lost upon selective removal of beta1 subunits (Messner and Catterall, 1986; Messner et al., 1986). Our results showing that coinjection of beta1 subunit mRNA significantly increases heart Na channel expression in Xenopus oocytes are consistent with the hypothesis that beta1 subunits are important for the biosynthesis, assembly, and stabilization of the cardiac Na channel in vivo.


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grant P01 HL44948 (to W. A. C.) and a Postdoctoral Research Fellowship from the American Heart Association, Washington Affiliate (to Y. Q.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address for L. L. I.: Dept. of Pharmacology, School of Medicine, University of Michigan, Ann Arbor, MI 48109.

Present address for J. C. R.: Howard Hughes Medical Institute, Depts. of Medicine and Physiology & Biophysics, School of Medicine, University of Iowa, Iowa City, IA 52242.

(^1)
The abbreviation used is: rH1, alpha subunit of rat cardiac Na channels.

(^2)
L. Isom, unpublished results.

(^3)
J. Rogers, T. Tanada, and W. A. Catterall, unpublished results.


ACKNOWLEDGEMENTS

We acknowledge Dr. David Ragsdale for teaching the oocyte injection and recording technique, and Alice B. Brownstein for expert technical assistance.


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