Abstract
Native smooth muscle L-type Ca(v)1.2 calcium channels have been shown to support a fraction of Ca(2+) currents with a window current that is close to resting potential. The smooth muscle L-type Ca(2+) channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca(v)1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Ca(v)1.2 channels. Here, we report a smooth muscle L-type Ca(v)1.2 calcium channel splice variant, Ca(v)1.2SM (1/8/9(*)/32/Delta33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca(v)1.2b clone (1/8/9(*)/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Ca(v)1.2CM clone (1a/8a/Delta9(*)/32/33) that is different from the established Ca(v)1.2a (1a/8a/Delta9(*)/31/33). Importantly, Ca(v)1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca(v)1.2b and cardiac Ca(v)1.2CM channels when currents were recorded in either 5 mM Ba(2+) or 1.8 mM Ca(2+) external solutions. This is the first time that a smooth muscle Ca(v)1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.
Highlights
This subpopulation of Cav1.2 calcium channels has not been identified
Alternative splicing serves to diversify Cav1.2 calcium channel biophysical properties and as such it is reasonable to test our first hypothesis as to whether a Cav1.2 splice variant may possess properties that are more similar to the subpopulation of channels that exhibit hyperpolarized window current
We showed that unexpectedly a single smooth muscle Cav1.2 splice variant, Cav1.2SM, underlies both a hyperpolarized window current that is reminiscent of the window current of a subpopulation of channels observed in smooth muscles [2,3,4] and a more potent inhibition by nifedipine in correlation with a hyperpolarized shift in steady-state inactivation potentials
Summary
Generation of Cardiac and Smooth Muscle Isoform of Cav1.2 Calcium Channels—The construction of human 77(⌬33) has been described previously [8]. It contains the combination of exons 1/8/⌬9*/32/⌬33. The steady-state inactivation data were fitted with a single Boltzmann equation: Irelative ϭ Imin ϩ (Imax Ϫ Imin)/(1 ϩ exp((V1/2 Ϫ V)/k), where Irelative is the normalized current; V1/2 is the potential for half-inactivation, and k is the slope value. The cells were activated by a 20-ms test pulse of variable voltage family from Ϫ60 to 120 mV, and tail currents were measured after repolarization to Ϫ50 mV for 10 ms. Statistical significance of differences between means was calculated with Student’s t test or one-way ANOVA. p Ͻ 0.05 is considered significant
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