L-type calcium channels are composed of a pore, _1c (Ca_V1.2), and accessory - and ₂-subunits. The -subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five ₂-subunits isoforms expressed in human heart (_2a-e) on the single L-type calcium channel current. These splice variants differ only by amino-terminal length and amino acid composition. Single-channel modulation by ₂-subunit isoforms was investigated in HEK293 cells expressing the recombinant L-type ion conducting pore. All ₂-subunits increased open probability, availability, and peak current with a highly consistent rank order (_2a2b>_2e2c>_2d). We show graded modulation of some transition rates within and between deep-closed and inactivated states. The extent of modulation correlates strongly with the length of amino-terminal domains. Two mutant ₂-subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.--Herzig, S., Khan, I. F. Y., Gründemann, D., Matthes, J., Ludwig, A., Michels, G., Hoppe, U. C., Chaudhuri, D., Schwartz, A., Yue, D. T., Hullin, R. Mechanism of Ca_v1.2 channel modulation by the amino terminus of cardiac ₂-subunits.