Ion-dependent Inactivation of Barium Current through L-type Calcium Channels

Address correspondence to Roman Shirokov, Department of Molecular Biophysics and Physiology, Rush University School of Medicine, 1750 W. Harrison St., Suite 1279JS, Chicago, IL 60612. Fax: 312-942-8711; E-mail: [email protected]

The tsA201 cell lines were a gift of Dr. M.M. Hosey (Northwestern University, Chicago, IL). The L-type Ca²⁺ channels cDNA sequences were gifts of Drs. E. Perez-Reyes (Loyola University, Maywood, IL) and M.M. Hosey. We thank Andy Chien, Tipu Puri and M.M. Hosey (Northwestern) for supplying the rabbit cardiomyocytes.

The horizontal transitions in schemei are voltage dependent, with rate constant k_RP,AP = a₁exp{(V − V₁)/2K − b[(V − V₁)/2K]²}, where V₁ is a transition voltage and K a steepness factor, the rate constant of the reverse transition is equal to k_RP,AP exp{(V₁ − V)/K}, and the rate constants between states RI and AI were calculated similarly, with a₂ and V₂ substituted for a₁ and V₁. a₁ = 100 s⁻¹, a₂ = 33 s⁻¹, V₁ = 0 mV, V₂ = −80 mV, K = 8 mV, and b = 0.1. The inactivation rate constants were: k_AP,AI = 0.15 s⁻¹, k_AI,AP = 0.05 s⁻¹. The rates of recovery from inactivation were set to satisfy kinetics of recovery, k_RP,RI + k_RI,RP = 20 s⁻¹, and microscopic reversibility, k_RI,RP/k_RP,RI = (k_AP,AI/k_AI,AP)exp [(V₁ − V₂)/2K ].

Dr. Ferreira's permanent address is Dept. Biofisica, Facultad de Medicina, Montevideo, Uruguay. Dr. Shirokov's permanent address is A.A. Bogomoletz Institute of Physiology, Kiev, Ukraine.

Parameters of schemeii were: k_CMe,C = 1 s⁻¹, k_UMe,U = 0.01 s⁻¹, k_C,U = 100 s⁻¹, k_U,C = 1,000 s⁻¹, k_UMe,CMe = 1,000 s⁻¹, k_CMe,UMe = 1 s⁻¹. k_C,CMe = k_U,UMe = 10⁶ M⁻¹s⁻¹ for Ca²⁺ and 10⁴ M⁻¹s⁻¹ for Ba²⁺.

Single channel currents in Ba²⁺ and Ca²⁺ were 0.6 and 0.3 pA, respectively, at 20 mV and saturating [Me²⁺]_e. At other voltages and [Me²⁺]_e = 10 mM, the amplitude of single channel current was calculated as XV{(exp[−V/12 mV])/(1 − exp[(−V/12 mV])} × {[Me²⁺]_e/([Me²⁺]_e + 14 mM)}, where X is 0.08 pA for Ca²⁺ and 0.16 pA for Ba²⁺.

Total current was calculated as N × P_o × i, where P_o is the probability that the three gates are in their permissive states. N was 125,000, corresponding to ≈25 channels/μm² in a cell of C_m = 50 pF. The channels generated both ionic and gating currents, in parallel with C_m and in series with resistance R_s = 5 MΩ. (The computer program can be obtained by E-mail requests to [email protected])