Voltage-dependent calcium channels in young and old human red cells

Author： Romero Pedro   Romero Eneida   Mateu Dania   Hernández Concepción   Fernández Irina  

Publisher： Humana Press, Inc

ISSN： 1085-9195

Source： Cell Biochemistry and Biophysics, Vol.46, Iss.3, 2006-10, pp. : 265-276

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Abstract

Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of α1C, subunit of high-voltage activated Ca channels (pan α1) and against conserved residues of α1C and α1E subunits. No specific reaction was detected with antibodies against conserved residues of α1A, α1B, or α1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan α1A or anti-α1E blots, whereas two bands (200 and 230 kDa) were detected by α1C exposure, Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers' under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl+5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15–50% reductions of 45Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Q-and N-type blockers also reduced Ca influx to various extents (25–60%). Old cells were generally insensitive to L-type but not to non-L-type, blockers. Raising external K to about 70–80 mM reduced by 50–100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non-L-type. The results, clearly demonstrate presence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non-L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed.

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