Novel functional properties of Ca(2+) channel beta subunits revealed by their expression in adult rat heart cells.
Recombinant adenoviruses were used to overexpress green fluorescent protein (GFP)-fused auxiliary Ca(2+) channel beta subunits (beta(1)-beta(4)) in cultured adult rat heart cells, to explore new dimensions of beta subunit functions in vivo. Distinct beta-GFP subunits distributed differentially between the surface sarcolemma, transverse elements, and nucleus in single heart cells. All beta-GFP subunits increased the native cardiac whole-cell L-type Ca(2+) channel current density, but produced distinctive effects on channel inactivation kinetics. The degree of enhancement of whole-cell current density was non-uniform between beta subunits, with a rank order of potency beta(2a) approximately equal to beta(4) > beta(1b) > beta(3). For each beta subunit, the increase in L-type current density was accompanied by a correlative increase in the maximal gating charge (Q(max)) moved with depolarization. However, beta subunits produced characteristic effects on single L-type channel gating, resulting in divergent effects on channel open probability (P(o)). Quantitative analysis and modelling of single-channel data provided a kinetic signature for each channel type. Spurred on by ambiguities regarding the molecular identity of the actual endogenous cardiac L-type channel beta subunit, we cloned a new rat beta(2) splice variant, beta(2b), from heart using 5' rapid amplification of cDNA ends (RACE) PCR. By contrast with beta(2a), expression of beta(2b) in heart cells yielded channels with a microscopic gating signature virtually identical to that of native unmodified channels. Our results provide novel insights into beta subunit functions that are unattainable in traditional heterologous expression studies, and also provide new perspectives on the molecular identity of the beta subunit component of cardiac L-type Ca(2+) channels. Overall, the work establishes a powerful experimental paradigm to explore novel functions of ion channel subunits in their native environments.
Colecraft
Henry M
HM
Program in Molecular and Cellular Systems Physiology, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Traylor Building, Room 710A, 720 Rutland Avenue, Baltimore, MD 21205, USA. hcolecra@bme.jhu.edu
Alseikhan
Badr
B
Takahashi
Shoji X
SX
Chaudhuri
Dipayan
D
Mittman
Scott
S
Yegnasubramanian
Vasan
V
Alvania
Rebecca S
RS
Johns
David C
DC
Marbán
Eduardo
E
Yue
David T
DT
D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000096:biosynthesis / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000187:drug effects; D017931:DNA Primers; D004594:Electrophysiology; D005822:Genetic Vectors; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials / Q000502:physiology; D008959:Models, Neurological; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
C484333:Cacnb3 protein, rat; C491529:Cacnb4 protein, rat; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D017931:DNA Primers; D011994:Recombinant Proteins