Distinctions in the molecular determinants of charged and neutral dihydropyridine block of L-type calcium channels

We investigated block of the alpha(1Cb) subunit of L-type calcium channels by dihydropyridines (DHPs) in which a permanently charged or neutral head g roup was linked to the active DHP moiety by a spacer chain containing ten m ethylene (-CH2) groups. We compared the sensitivity of channel modulation b y the charged (DHPch) and neutral (DHPn) forms to specific ct,,, mutations in domains IIIS5, IIIS6, and IVS6, which had previously been shown to reduc e channel modulation by the neutral DHP (+)-isradipine. The effects of thes e mutations were studied on channel block recorded from polarized (-80 mV) and depolarized (-40 mV) holding potentials (HPs), We found that channel bl ock by DHPn was markedly reduced at both HPs by each mutation studied. In c ontrast, channel block by DHPch was only modestly reduced by mutations in I IIS6 and IVS6 for block from either -40 mV or -80 mV. Replacement of IIIS5 Thr1061 by Tyr, which abolished block by DHPn in an HP-independent manner, had little effect on channel block by DHPch recorded from -40 mV, However, this mutation markedly reduced DHPch block of currents recorded from a -80 mV HP. Inhibition of current by DHPch was not markedly use-dependent, in co ntrast with block by verapamil, another charged calcium channel blocker, Th ese results suggest that the presence of a permanently charged head group r estricts the access of the attached DHP moiety to a subset of interaction r esidues on the alpha(1C) subunit in a voltage-dependent manner. Furthermore , these restricted interactions confer distinct functional properties upon the charged DHP molecules.