MOLECULAR FUNCTION OF CHIMERIC Ca-Na IONIC CHANNELS

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BACKGROUND and OBJECTIVERecent electrophysiological studies combined with molecular biology provided evidence that the kinetics of the L-type Ca^<2+> channel derives largely from the structure of the pore-forming subunit of the channel (alpha_<1c> subunit), however, the molecular events underlying the formation of subconductance levels are still under investigation. To understand the nature and structural requirements for subconductance, we have expressed cardiac alpha_<1c> L-type Ca^<2+> channels with or without coexpression of the beta_2 subunit in COS cells or in Chinese Hamster Fibroblast (CHW) cells to determine if alpha_<1c> channels and alpha_<1c>/beta_2 channels display different single channel subconductance levels.METHODSTransfection of COS- and CHW-cells : COS cells transiently transfected with cDNA encoding the alpha_1 subunit of the cardiac L-type calcium channel (alpha_<1c>), and CHW cells stably transfected with alpha_1 subunit alone or with alpha_<1c> plus cardiac beta … More subunits (alpha_<1c>/beta_2), were maintained in cell culture (see below). Electrophysiology : Small (conventional) patch pipettes with resistances between 4-5 MOMEGA allowed recording of alpha_<1c>/beta_2 channels. Large (macropatch) pipettes with resistances between 0.2-0.3 MOMEGA allowed recording of alpha_<1c> channels. Patches were depolarized for 195 ms at every 0.5-1 seconds or for 395ms at every 5 seconds from a holding potential (V_H) of -80mV.In ramp clamp experiments, slope ratio of -0.1V/sec was applied at every 3 seconds.RESULTS(1) Structural requirements for the formation of subconducting state (s) of the L-type Ca^<2+> channel were studied in cardiac alpha_<1c> channels in COS- and CHW-cells with or without coexpression of beta_2-subunit.(2) Similar four conductance levels were detected in alpha_<1c> alone and in alpha_<1c>/beta_2 channels : 3.4<plus-minus>0.2pS,6.4<plus-minus>0.2pS,14.5<plus-minus>0.2pS and 22.2<plus-minus>0.2pS(3) Existence of pore-forming alpha_<1c> subunit alone fulfillls the condition to form multiple conducting states.(4) Coexpression of beta_2-subunit leads to a higher probability of channel openings at three subconductance levels as well as full conducting level of 22 pS,but may not be prerequisite for production of sublevel openings in cardiac L-type Ca^<2+> channels. Less