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Tyrosine kinase inhibitors and ATP modulate the conversion of smooth muscle L-type Ca²⁺ channels toward a second open state

Shinsuke Nakayama^*,1, Yasushi Ito, Shinji Sato, Atsushi Kamijo^*, Hong-Nian Liu^* and Shunichi Kajioka^*,2

Department of

^* Cell Physiology and

Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan

¹ Correspondence: Department of Cell Physiology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: h44673a{at}nucc.cc.nagoya-u.ac.jp

SPECIFIC AIMS

Properties of smooth and cardiac L-type Ca²⁺ channels differ prominently in several physiological aspects, although these channels are splice products with 95% homology in amino acid sequence. Here, we show that tyrosine kinase inhibitors and ATP alter the kinetics of smooth muscle Ca²⁺ channels, mimicking that of cardiac Ca²⁺ channels seen under normal conditions.

PRINCIPAL FINDINGS

1. A second open state evoked in a voltage-dependent manner
Relatively large depolarizations convert the smooth muscle channel conformation from the normal (O₁) to a second open state (O₂) in which Ca²⁺ channels inactivate only very slowly or not at all during depolarization, and deactivate slowly on repolarization.

In enzymatically isolated detrusor smooth muscle cells, Ca ²⁺ channel currents were evoked by test steps (0 mV, 100 ms) with and without a preconditioning step (+80 mV, 4 s) (i.e., paired pulse protocol). Under normal conditions, the peak amplitude of the conditioned test current (I_c) was 1.4 times larger than that of the unconditioned test current (I_u). Slowly deactivating tail currents (_tail=10 ms) were observed after preconditioning depolarization.

The recovery of the availability of Ca²⁺ channels and slow-tail current seen after a preconditioning step depolarization at +80 mV are considered to reflect the O₂ state that evoked by a voltage-dependent process.

2. Different effects of genistein on conditioned and unconditioned Ca²⁺ channel currents
Genistein, a soybean isoflavone, is known to selectively inhibit protein tyrosine kinase (PTK). We examined the effects of this drug on Ca²⁺ channel currents evoked by the paired pulse protocol. Application of 10 µM genistein in the extracellular medium gradually reduced both conditioned (I_c) and unconditioned test currents (I_u). The suppression was more pronounced in I_c, thereby the I_c/I_u ratio decreased. The decay time constant of the slow-tail current did not change.

Correlation of the current density of test inward current and the I_c/I_u ratio was analyzed. In the presence of genistein, these two parameters were smaller than those obtained under normal conditions.

3. Inactivation property in the presence of genistein
In the presence of 10 µM genistein, the voltage-dependent inactivation property was examined over a wide voltage range (–100 to +80 mV, 4 s). The inactivation curve was clearly U-shaped, with a maximal degree of inactivation at 0 mV. However, the recovery of the test current amplitude at +80 mV (I₊₈₀) was significantly smaller compared to I₊₈₀ recorded under normal conditions.

4. Effects of genistin, an inactive analog of genistein
During the application of genistin in the extracellular medium, I_c and I_u only slowly decreased by similar degrees, and the I_c/I_u ratio remained at around the control concentration.

5. Intracellular application of genistein and tyrphostin-47
Genistein was next applied through the patch pipette. When the patch pipette contained 1 mM ATP like the experiments examining the effects of extracellular applications, the inclusion of genistein (10–30 µM) in the patch pipette did not show much effect on Ca²⁺ channel currents evoked by the paired pulse protocol.

Genistein inhibits PTK by competing with ATP. Removal of ATP yielded dramatic changes in the effects of intracellular application of genistein. During the experiment, both I_c and I_u decreased, but the suppression of I_c was more pronounced. As a result, the I_c/I_u ratio was significantly decreased, as seen in extracellular applications. Intracellular application of tyrphostin-47, another PTK inhibitor, caused a similar suppression of Ca²⁺ channel currents evoked by the paired pulse protocol, i.e., significant decrease in I_c/I_u (Fig. 1 ).

View larger version (16K): [in this window] [in a new window]   Figure 1. Effects of tyrphostin-47 application via the patch pipette containing no ATP. A) Whole cell currents evoked by the paired pulse protocol. Dotted lines indicate zero current levels. The left calibration bar for (a) and (b); right for (c) and (d). The patch pipette contained 30 µM tyrphostin-47, but no ATP. B) The time courses of changes in Ic (squares), Iu (circles), and the Ic/Iu ratio (triangles). Conditioned and unconditioned experiments are shown in red and blue, respectively. The Ic/Iu ratio is shown in green.

ATP is involved in any kinase activity. When the cells were dialyzed with pipette solution containing neither ATP nor PTK inhibitors, both I_c and I_u gradually decreased. The decrease in I_c/I_u was, however, attenuated in the absence of PTK inhibitors.

6. Intracellular application of PP2
PP2 is known to inhibit the PTK activity of src in a noncompetitive manner against ATP. Even in the presence of ATP, intracellular application of PP2 significantly reduced the I_c/I_u ratio, accompanied by acceleration of I_u inactivation.

7. Kinetic model
The voltage-dependent U-shaped inactivation and slow deactivation properties can be described by a minimum kinetic scheme with one closed state (C), two open states (O₁, O₂), and two inactivated states (I₀, I₁) linked to C and O₁, respectively. The rate constant k₁₄ for the O₁ to I₁ transition could be responsible for the characteristic features seen during application of PTK inhibitors (Fig. 2 A). As the rate constant k₁₄ for the O₁ to I₁ transition increases, the availability of Ca²⁺ channels at positive conditioning potentials decreases (Fig. 2B ), accompanied by progressive increases in the inactivation rate of the test current without a conditioning depolarization (Fig. 2C ).

View larger version (41K): [in this window] [in a new window]   Figure 2. Kinetic model for smooth muscle Ca2+ channels. The kinetic scheme used for the computer simulation is shown in A. (Abbreviations, see text.) The k(s) represent the rate constants of transition. A) Normal smooth muscle mode. antibody: PTK-impaired mode, where the transition rate from O1 to I1 is increased (k14*). This progressively attenuates the voltage-dependent conversion of Ca2+ channels toward O2, mimicking cardiac Ca2+ channel kinetics under normal conditions. In B), Percentage of available Ca2+ channels (C+O1+O2) at the end of conditioning step (4 s) is plotted against the conditioning potential. Circles represent available channels under normal conditions, whereas squares and triangles represent those in case of x5 and x15 k14, respectively. C) Computer calculation for Ca2+ channel current evoked by a step depolarization to 0 mV. Normal condition (a); k14* = x5 (b); k14* = x15 (c). As the k14 rate increases, the recovery phase of availability (at positive conditioning potentials) is attenuated, and simultaneously, inactivation of Ca2+ channel current evoked by a step depolarization is accelerated.

CONCLUSIONS AND SIGNIFICANCE

A fundamental question remains why smooth and cardiac L-type Ca²⁺ channels, sharing 95% amino acid sequence, play prominently different roles during sympathetic stimulation. It is well known that cardiac muscle L-type Ca²⁺ channels play a central role in the inotropic action of sympathetic nerves through stimulation of ßbeta;-adrenoceptors and initiation of the cyclic AMP cascade, resulting in phosphorylation of the channel protein. On the other hand, sympathetic stimulation relaxes most smooth muscles, including detrusor, via activation of ßbeta;-adrenoceptors, and smooth muscle L-type Ca²⁺ channels are not sufficiently enhanced to alter physiological function especially in terms of cyclic AMP cascade.

The present patch-clamp experiments have revealed that the genistein-induced suppression of L-type Ca²⁺ current is much more pronounced after a preconditioning depolarization. This unique effect of genistein is ascribed to tyrosine-kinase-related mechanisms or factors for the following reasons. First, application of tyrphostin-47, another tyrosine kinase inhibitor, reproduced this voltage-dependent inhibitory effect but genistin, inactive analog of genistein did not. Secondly, irrespective of extracellular or intracellular application, tyrosine kinase inhibitors caused similar changes in the I_c/I_u ratio. Furthermore, intracellular dialysis with a pipette solution containing no ATP synergically potentiated the inhibitory action of genistein. This antagonistic effect of ATP on PTK inhibition is a feature of genistein and structurally similar PTK inhibitors.

From the patch-clamp measurements and computer simulation, it is concluded that mechanism(s) relating to ATP and PTK confer the noninactivating O₂ state to smooth muscle L-type Ca²⁺ channels, in addition to increasing their availability. Acceleration of the O₁ to I₁ transition could systematically explain the effects of PTK inhibitors. It is also speculated that this mechanism may provide an important link of channel kinetics between smooth and cardiac Ca²⁺ channels, i.e., even under normal conditions, smooth muscle Ca²⁺ channels are set in a channel mode, similar to cardiac L-type Ca²⁺ channels on ßbeta;-adrenoreceptor stimulation. Furthermore, the present study opens the possibility that Ca²⁺ influx through the noninactivating O₂ state might play an important role in cell growth and cell-matrix interaction, because PTK-related mechanisms or factors underlie this open state. To elucidate the detailed control and functional diversity of L-type Ca²⁺ channels, further investigation of the PTK-related modulation of the noninactivating O₂ state, including the roles of ßbeta;- and other auxiliary subunits is required.

FOOTNOTES

² Present address: Oita University School of Medicine, Oita 879-5593, Japan.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5049fje

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-5049fjev1 20/9/1492    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nakayama, S. Articles by Kajioka, S. Search for Related Content PubMed PubMed Citation Articles by Nakayama, S. Articles by Kajioka, S.