Functional effects of point mutations in the S4 motif of cone cyclic GMP-gated ion channels

MP FAILLACE AND JI KORENBROT

Tucson, ARIZONA

Workshop; The Federation of American Societies for Experimental Biology. (FASEB) Summer Research Conferences, 2003. The Biology and Chemistry of Vision.; 2003

The Federation of American Societies for Experimental Biology. (FASEB)

  Cyclic GMP-gated ion channels (CNG) in photoreceptors are not gated by membrane voltage, yet they exhibit a number of voltage-dependent behaviors: in rods the open channel lifetime is higher at depolarized than hyperpolarized voltages. In both rods and cones, the binding affinity for cGMP is slightly lower at depolarized (+50 mV) than hyperpolarized (-50 mV) membrane potentials and the relative ion permeability between Na+ and Ca2+ (PCa/PNa) is also voltage-dependent.  CNG channels share with voltage-gated channels (VG) (whether K+, Na+ or Ca2+) a structural motif named “S4” that consists of 4 sequentially repeated amino acid triplets arginine-XX (or lysine) in the 4th transmembrane helix. The S4 motif function as a voltage-sensor in VG channels: changes in membrane voltage cause a translational movement of the S4 domain that is linked to channel opening.  The specific function of S4 in CNG channels is unknown. To determine a possible function of S4 in CNG, we investigated the fraction of the cGMP-gated current carried by calcium (Pf) in CNGA3 cone channels a subunits heterogeneously expressed in TSA-201 cells.  To determine Pf, we measured simultaneously membrane current and cytoplasmic Ca2+ concentration in single cells in which we activated CNG channels with a caged cGMP analog (caged 8-pCPT-cGMP).  Just as we have previously observed in intact cone photoreceptors, wt CNG3 demonstrated an independence of Pf on membrane voltage over the physiological voltage range (-85 to –35 mV).  This behavior is inconsistent with electrodiffusion theory and reflects a voltage-dependent change in PCa/PNa.  To explore a possible relevance of S4 to this behavior, we mutated either one by one or all at once the four regularly spaced basic residues of the S4 segment. The basic amino acids arginine (R293, 296, 302) or lysine (K299) were mutated to glutamine. We also changed R296 to cysteine and R302 to tryptophan, mutations identified as associated with a congenital human achromatopsia.  Electrophysiological assays revealed that each and every one of the mutations resulted in a failure to express functional channels in the cells membrane surface.  We are investigating whether the functional failure arises from lack of protein expression or errors in protein cellular processing (folding, transport and/or sorting).  Our results may suggest that S4 is conserved among various channel types not because of its potential role as a voltage-sensor, but because of its role in channel protein processing.