Abstract
L-type Ca(2+) channels are heteromultimeric and finely tuned by auxiliary subunits in different tissues and regions. Among auxiliary subunits, beta subunit has been shown to play important roles in many functional aspects of Ca(2+) channel. Rat heart was reported to specifically express beta(2a) subunit. However, the slow inactivation rates of Ca(2+) currents recorded from recombinant Ca(2+) channels with the beta(2a) subunit, and the reported inability to detect beta(2a) subunit in rabbit heart by reverse transcription-PCR analysis raise the possibility of the existence of other beta subunits. We cloned a splice variant of beta(2) subunit from rat heart, using rapid amplification of cDNA 5' ends. The splice variant is highly similar to human beta(2c) subunit that was cloned from human ventricle. Northern blot analysis detected the rat beta(2c) subunit abundantly in rat heart and brain. The deduced amino acid sequence of the beta(2c) subunit was different from that of the beta(2a) subunit only in the N-terminal region. When the beta(2c) subunit was expressed along with alpha(1c) and alpha(2)delta subunits in baby hamster kidney cells, the inactivation rates were comparable with those from native cardiac myocytes, although those with the beta(2a) subunit were slow. Taken together, these observations suggest that the beta(2c) subunit is a functional beta(2) subunit expressed in heart and that the short N-terminal region plays a major role in modifying inactivation kinetics.
Highlights
L-type Ca2ϩ channel plays an important role in shaping the action potential of cardiac myocytes and is a major pathway for extracellular Ca2ϩ entry into cardiomyocytes [1]
When the 2c subunit was expressed in BHK cells along with a pore-forming ␣1c and other auxiliary ␣2␦ subunits cloned from rat heart, the inactivation rate was comparable with that from native cardiac myocytes
As shown in lanes 3 and 4 of Fig. 1B, only primer_3 and primer_2 detected the 2 subunit, which indicated that the N-terminal region of the 2 subunit expressed in rat heart was different from the reported 2a sequence cloned from rat brain
Summary
Preparation of Single Cardiac Myocytes—Single ventricular myocytes were enzymatically isolated from the ventricle of rat hearts as described previously [16]. The hearts were removed from rats, following anesthetic with pentobarbital, and perfused in a Langendorff apparatus with 0.02– 0.04% collagenase (Wako Pure Chemical Industries, Osaka, Japan) dissolved in nominally Ca2ϩ-free Tyrode solution. After 30 min of digestion, the left ventricle was rinsed with Kraftbruhe (KB) solution [17], cut into small pieces, and shaken to separate cells. TotalPrimer1104f TotalPrimer1104r ␣2primer909f ␣2primer909r total-beta-2-primer414f total-beta-2-primer414r 2primer302f 2primer302r 2primer_for_5Ј-RACE604r1 2primer_for_5Ј-RACE604r2 2primer_for_5Ј-RACE604r3 brain-beta-5Ј G-1208f brain-beta-5Ј G-1208r heart beta-5Ј G-1208f heart beta-5Ј G-1208r tbeta2-p-308f tbeta2-p-308R
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