Zimmer T and Benndorf K (2002), The human heart and rat brain IIA Na+ channels ...

XB-ART-6145

J Gen Physiol 2002 Dec 01;1206:887-95. doi: 10.1085/jgp.20028703.

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The human heart and rat brain IIA Na+ channels interact with different molecular regions of the beta1 subunit.

Zimmer T , Benndorf K .

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The alpha subunit of voltage-gated Na(+) channels of brain, skeletal muscle, and cardiomyocytes is functionally modulated by the accessory beta(1), but not the beta(2) subunit. In the present study, we used beta(1)/beta(2) chimeras to identify molecular regions within the beta(1) subunit that are responsible for both the increase of the current density and the acceleration of recovery from inactivation of the human heart Na(+) channel (hH1). The channels were expressed in Xenopus oocytes. As a control, we coexpressed the beta(1)/beta(2) chimeras with rat brain IIA channels. In agreement with previous studies, the beta(1) extracellular domain sufficed to modulate IIA channel function. In contrast to this, the extracellular domain of the beta(1) subunit alone was ineffective to modulate hH1. Instead, the putative membrane anchor plus either the intracellular or the extracellular domain of the beta(1) subunit was required. An exchange of the beta(1) membrane anchor by the corresponding beta(2) subunit region almost completely abolished the effects of the beta(1) subunit on hH1, suggesting that the beta(1) membrane anchor plays a crucial role for the modulation of the cardiac Na(+) channel isoform. It is concluded that the beta(1) subunit modulates the cardiac and the neuronal channel isoforms by different molecular interactions: hH1 channels via the membrane anchor plus additional intracellular or extracellular regions, and IIA channels via the extracellular region only.

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	Figure 1. . Modulation of hH1 and IIA Na+ channels by the Î²1 subunit. (A) Representative current traces for hH1 and hH1/Î²1 channels at the test potentials â10 and â30 mV. The Ïh values of hH1 versus hH1/Î²1 channels were statistically indistinguishable (â30 mV: Ïh = 2.21 Â± 0.18 for hH1, Ïh = 2.01 Â± 0.43 for hH1/Î²1 [P = 0.64]; â10 mV: Ïh = 1.25 Â± 0.07 for hH1, Ïh = 1.12 Â± 0.08 for hH1/Î²1 [P = 0.23]). Number of experiments: n = 11 for hH1, n = 6 for hH1/Î²1. Calibration bars = 4 ms, at â30 mV: 0.25 Î¼A for hH1, 0.62 Î¼A for hH1/Î²1; at â10 mV: 0.45 Î¼A for hH1, 1.06 Î¼A for hH1/Î²1. (B) Effect of the Î²1 subunit on the hH1 peak current amplitude in Xenopus oocytes (P < 0.001). Currents were measured 3 d after injection at the test potential of â25 mV. Measurements were performed in seven different batches of oocytes. Data from a single batch of oocytes were normalized with respect to the mean current of hH1-injected oocytes (n = 63 for hH1, n = 50 for hH1/Î²1, and n = 52 for hH1/Î²2). (C) Time course of recovery from inactivation of hH1 channels. The respective voltage protocol is shown in the inset (n = 32 for hH1, n = 38 for hH1/Î²1, and n = 21 for hH1/Î²2). (D) Effect of the Î²1 subunit on the inactivation time course of rat brain IIA Na+ currents. The Na+ currents were elicited by a test pulse to â10 mV, and normalized with respect to the peak current. Calibration bars = 5 ms, 0.9 Î¼A for IIA, 0.8 Î¼A for IIA/Î²1, and 0.7 Î¼A for IIA/Î²2. Statistically, the inactivation time constant Ïh was not different for IIA and IIA/Î²2 channels at the applied test pulses (unpublished data). (E) Effect of the Î²1 subunit on the IIA peak current amplitude (P < 0.001). Currents were measured 3 d after injection at the test potential of â10 mV (n = 13 for IIA, n = 13 for IIA/Î²1 and n = 13 for IIA/Î²2). (F) Time course of recovery from inactivation of IIA channels. Currents were elicited by the same voltage protocol as indicated in C, except that a test pulse to â10 mV was used (n = 7 for IIA, n = 7 for IIA/Î²1 and n = 6 for IIA/Î²2). Bars indicate SEM.
	Figure 2. . Structure of the chimeras between the Na+ channel Î²1 and Î²2 subunits used in this study. The corresponding terminal amino acids of the Î²1 (white boxes) and Î²2 (gray boxes) subunit regions are indicated. The assumed topology of both subunits in the plasma membrane is shown below the cartoons of the chimeras.
	Figure 3. . Modulatory effect of chimeras Î²122 and Î²211 on hH1 and IIA channels. (A) Schematic representation of the domain structures of both chimeras. (B) Relative current amplitudes (test pulse to â25 mV; P < 0.001; n = 55 for hH1, n = 37 for hH1/Î²1, n = 38 for hH1/Î²122, and n = 50 for hH1/Î²211). (C) Time course of recovery from inactivation of hH1 channels (n = 7 for hH1, n = 6 for hH1/Î²1, n = 5 for hH1/Î²122, and n = 6 for hH1/Î²211). (D) Effect of Î²122 on the inactivation time course of rat brain IIA Na+ currents (test pulse: â10 mV). Calibration bars = 5 ms, 0.25 Î¼A for IIA, 0.52 Î¼A for IIA/Î²1, 0.27 Î¼A for IIA/Î²122, and 0.21 Î¼A for IIA/Î²211. (E) Effect of Î²122 on the IIA peak current amplitude (test pulse to â10 mV; P < 0.001; n = 19 for IIA, n = 13 for IIA/Î²1, n = 19 for IIA/Î²122, and n = 17 for IIA/Î²211). (F) Time course of recovery from inactivation of IIA channels. For voltage protocol, see legend to Fig. 1 (n = 13 for IIA, n = 12 for IIA/Î²1 and n = 12 for IIA/Î²122, and n = 12 for IIA/Î²211). Bars indicate SEM.
	Figure 4. . Coexpression of chimeras Î²121 and Î²221 with hH1 and IIA channels. (A) Schematic representation of the domain structures of both chimeras. (B) Relative current amplitudes (test pulse to â25 mV; P < 0.001; n = 29 for hH1, n = 17 for hH1/Î²1, n = 28 for hH1/Î²121, and n = 21 for hH1/Î²221). (C) Time course of recovery from inactivation of hH1 channels (n = 21 for hH1, n = 17 for hH1/Î²1, n = 12 for hH1/Î²121, and n = 19 for hH1/Î²221). (D) Effect of Î²121 on the time course of inactivation of rat brain IIA Na+ currents (test pulse to â10 mV). Calibration bars = 5 ms, 0.93 Î¼A for IIA, 0.90 Î¼A for IIA/Î²1, 0.62 Î¼A for IIA/Î²121, and 0.90 Î¼A for IIA/Î²221. (E) Effect of Î²121 and Î²221 on the IIA peak current amplitude (test pulse to â10 mV; P < 0.001; n = 15 for IIA, n = 9 for IIA/Î²1, n = 12 for IIA/Î²121, and n = 19 for IIA/Î²221). (F) Time course of recovery from inactivation of IIA channels (n = 13 for IIA, n = 9 for IIA/Î²1 and n = 5 for IIA/Î²121, and n = 13 for IIA/Î²221). Bars indicate SEM.
	Figure 5. . Coexpression of chimera Î²212 and Î²21Î with hH1 channels. (A) Schematic representation of the domain structure of Î²212 and Î²21Î. (B) Relative current amplitudes (test pulse to â25 mV; *P < 0.001; n = 17 for hH1, n = 10 for hH1/Î²1, n = 21 for hH1/Î²212, and n = 11 for Î²21Î). (C) Time course of recovery from inactivation of hH1 channels (n = 10 for hH1, n = 4 for hH1/Î²1, n = 11 for hH1/Î²212, and n = 7 for Î²21Î). Bars indicate SEM.
	Figure 6. . Coexpression of Î²11Î and Î²12Î with hH1 and IIA channels. (A) Schematic representation of the domain structures of both deletion variants. (B) Relative current amplitudes (test pulse to â25 mV; P < 0.001; n = 38 for hH1, n = 38 for hH1/Î²1, n = 30 for hH1/Î²11Î, and n = 23 for hH1/Î²12Î). (C) Time course of recovery from inactivation of hH1 channels (n = 23 for hH1, n = 21 for hH1/Î²1, n = 19 for hH1/Î²11Î, and n = 20 for hH1/Î²12Î). (D) Effect of Î²11Î and Î²12Î on the inactivation time course of rat brain IIA Na+ currents (test pulse to â10 mV). Calibration bars = 5 ms, 0.92 Î¼A for IIA, 0.76 Î¼A for IIA/Î²1, 0.80 Î¼A for IIA/Î²11Î, and 0.28 Î¼A for IIA/Î²12Î. (E) Effect of Î²11Î and Î²12Î on the IIA peak current amplitude (test pulse to â10 mV; P < 0.001; n = 16 for IIA, n = 14 for IIA/Î²1, n = 21 for IIA/Î²11Î, and n = 9 for IIA/Î²12Î). (F) Time course of recovery from inactivation of IIA channels (n = 13 for IIA, n = 13 for IIA/Î²1, n = 16 for IIA/Î²11Î, and n = 10 for IIA/Î²12Î). Bars indicate SEM.

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	Figure 1. . Modulation of hH1 and IIA Na+ channels by the Î²1 subunit. (A) Representative current traces for hH1 and hH1/Î²1 channels at the test potentials â10 and â30 mV. The Ïh values of hH1 versus hH1/Î²1 channels were statistically indistinguishable (â30 mV: Ïh = 2.21 Â± 0.18 for hH1, Ïh = 2.01 Â± 0.43 for hH1/Î²1 [P = 0.64]; â10 mV: Ïh = 1.25 Â± 0.07 for hH1, Ïh = 1.12 Â± 0.08 for hH1/Î²1 [P = 0.23]). Number of experiments: n = 11 for hH1, n = 6 for hH1/Î²1. Calibration bars = 4 ms, at â30 mV: 0.25 Î¼A for hH1, 0.62 Î¼A for hH1/Î²1; at â10 mV: 0.45 Î¼A for hH1, 1.06 Î¼A for hH1/Î²1. (B) Effect of the Î²1 subunit on the hH1 peak current amplitude in Xenopus oocytes (P < 0.001). Currents were measured 3 d after injection at the test potential of â25 mV. Measurements were performed in seven different batches of oocytes. Data from a single batch of oocytes were normalized with respect to the mean current of hH1-injected oocytes (n = 63 for hH1, n = 50 for hH1/Î²1, and n = 52 for hH1/Î²2). (C) Time course of recovery from inactivation of hH1 channels. The respective voltage protocol is shown in the inset (n = 32 for hH1, n = 38 for hH1/Î²1, and n = 21 for hH1/Î²2). (D) Effect of the Î²1 subunit on the inactivation time course of rat brain IIA Na+ currents. The Na+ currents were elicited by a test pulse to â10 mV, and normalized with respect to the peak current. Calibration bars = 5 ms, 0.9 Î¼A for IIA, 0.8 Î¼A for IIA/Î²1, and 0.7 Î¼A for IIA/Î²2. Statistically, the inactivation time constant Ïh was not different for IIA and IIA/Î²2 channels at the applied test pulses (unpublished data). (E) Effect of the Î²1 subunit on the IIA peak current amplitude (P < 0.001). Currents were measured 3 d after injection at the test potential of â10 mV (n = 13 for IIA, n = 13 for IIA/Î²1 and n = 13 for IIA/Î²2). (F) Time course of recovery from inactivation of IIA channels. Currents were elicited by the same voltage protocol as indicated in C, except that a test pulse to â10 mV was used (n = 7 for IIA, n = 7 for IIA/Î²1 and n = 6 for IIA/Î²2). Bars indicate SEM.
	Figure 2. . Structure of the chimeras between the Na+ channel Î²1 and Î²2 subunits used in this study. The corresponding terminal amino acids of the Î²1 (white boxes) and Î²2 (gray boxes) subunit regions are indicated. The assumed topology of both subunits in the plasma membrane is shown below the cartoons of the chimeras.
	Figure 3. . Modulatory effect of chimeras Î²122 and Î²211 on hH1 and IIA channels. (A) Schematic representation of the domain structures of both chimeras. (B) Relative current amplitudes (test pulse to â25 mV; P < 0.001; n = 55 for hH1, n = 37 for hH1/Î²1, n = 38 for hH1/Î²122, and n = 50 for hH1/Î²211). (C) Time course of recovery from inactivation of hH1 channels (n = 7 for hH1, n = 6 for hH1/Î²1, n = 5 for hH1/Î²122, and n = 6 for hH1/Î²211). (D) Effect of Î²122 on the inactivation time course of rat brain IIA Na+ currents (test pulse: â10 mV). Calibration bars = 5 ms, 0.25 Î¼A for IIA, 0.52 Î¼A for IIA/Î²1, 0.27 Î¼A for IIA/Î²122, and 0.21 Î¼A for IIA/Î²211. (E) Effect of Î²122 on the IIA peak current amplitude (test pulse to â10 mV; P < 0.001; n = 19 for IIA, n = 13 for IIA/Î²1, n = 19 for IIA/Î²122, and n = 17 for IIA/Î²211). (F) Time course of recovery from inactivation of IIA channels. For voltage protocol, see legend to Fig. 1 (n = 13 for IIA, n = 12 for IIA/Î²1 and n = 12 for IIA/Î²122, and n = 12 for IIA/Î²211). Bars indicate SEM.
	Figure 4. . Coexpression of chimeras Î²121 and Î²221 with hH1 and IIA channels. (A) Schematic representation of the domain structures of both chimeras. (B) Relative current amplitudes (test pulse to â25 mV; P < 0.001; n = 29 for hH1, n = 17 for hH1/Î²1, n = 28 for hH1/Î²121, and n = 21 for hH1/Î²221). (C) Time course of recovery from inactivation of hH1 channels (n = 21 for hH1, n = 17 for hH1/Î²1, n = 12 for hH1/Î²121, and n = 19 for hH1/Î²221). (D) Effect of Î²121 on the time course of inactivation of rat brain IIA Na+ currents (test pulse to â10 mV). Calibration bars = 5 ms, 0.93 Î¼A for IIA, 0.90 Î¼A for IIA/Î²1, 0.62 Î¼A for IIA/Î²121, and 0.90 Î¼A for IIA/Î²221. (E) Effect of Î²121 and Î²221 on the IIA peak current amplitude (test pulse to â10 mV; P < 0.001; n = 15 for IIA, n = 9 for IIA/Î²1, n = 12 for IIA/Î²121, and n = 19 for IIA/Î²221). (F) Time course of recovery from inactivation of IIA channels (n = 13 for IIA, n = 9 for IIA/Î²1 and n = 5 for IIA/Î²121, and n = 13 for IIA/Î²221). Bars indicate SEM.
	Figure 5. . Coexpression of chimera Î²212 and Î²21Î with hH1 channels. (A) Schematic representation of the domain structure of Î²212 and Î²21Î. (B) Relative current amplitudes (test pulse to â25 mV; *P < 0.001; n = 17 for hH1, n = 10 for hH1/Î²1, n = 21 for hH1/Î²212, and n = 11 for Î²21Î). (C) Time course of recovery from inactivation of hH1 channels (n = 10 for hH1, n = 4 for hH1/Î²1, n = 11 for hH1/Î²212, and n = 7 for Î²21Î). Bars indicate SEM.
	Figure 6. . Coexpression of Î²11Î and Î²12Î with hH1 and IIA channels. (A) Schematic representation of the domain structures of both deletion variants. (B) Relative current amplitudes (test pulse to â25 mV; P < 0.001; n = 38 for hH1, n = 38 for hH1/Î²1, n = 30 for hH1/Î²11Î, and n = 23 for hH1/Î²12Î). (C) Time course of recovery from inactivation of hH1 channels (n = 23 for hH1, n = 21 for hH1/Î²1, n = 19 for hH1/Î²11Î, and n = 20 for hH1/Î²12Î). (D) Effect of Î²11Î and Î²12Î on the inactivation time course of rat brain IIA Na+ currents (test pulse to â10 mV). Calibration bars = 5 ms, 0.92 Î¼A for IIA, 0.76 Î¼A for IIA/Î²1, 0.80 Î¼A for IIA/Î²11Î, and 0.28 Î¼A for IIA/Î²12Î. (E) Effect of Î²11Î and Î²12Î on the IIA peak current amplitude (test pulse to â10 mV; P < 0.001; n = 16 for IIA, n = 14 for IIA/Î²1, n = 21 for IIA/Î²11Î, and n = 9 for IIA/Î²12Î). (F) Time course of recovery from inactivation of IIA channels (n = 13 for IIA, n = 13 for IIA/Î²1, n = 16 for IIA/Î²11Î, and n = 10 for IIA/Î²12Î). Bars indicate SEM.