Nicotinic acetylcholine receptor channels are influenced by the physical state of their membrane environment.
We investigated the effect of the physical state of the cell membrane on the activity of the nicotinic acetylcholine receptor (AChR) in various clonal cell lines transfected with the cDNAs of embryonic or adult AChR by measuring single-channel properties and some membrane physicochemical properties as a function of temperature. Unitary conductance and channel closing rate, alpha, had Q(10) values of 1.2 and 2.2, respectively. Using Eyring's transition state theory, it was calculated that both embryonic and adult-type AChR had relatively low thermal sensitivity of ionic conductance and activation energy (E(a) of 3.0-5.0 kcal-mol(-1) at 20 degrees C), indicating that once the AChR channel opens, ion movement is dominated by diffusional processes. Channel closure exhibited higher energy requirements, with E(a) values of about 13 kcal-mol(-1). This process appears to be more endothermic (higher delta H(a) values) than ion permeation, and it is plausible that the energy acquired by the system can be used in the maintenance of its degree of order, as revealed by the delta S(a) 0 calculated for channel closure. The influence of the membrane environment on AChR function is reinforced by the observation that the conductance of the same, embryonic-type AChR protein, expressed in qualitatively different cellular lipid environments, appeared to have different energetic requirements. A correlation between the electrophysiological and thermodynamic parameters of the AChR and physicochemical properties of the membrane bilayer in which the protein is embedded could be established using measurements of the so-called generalized polarization (GP) of the lipophilic probe laurdan. Both embryonic and adult AChR exhibited a higher GP and a higher sensitivity to temperature-dependent changes in GP when heterologously expressed in stable form in Chinese hamster ovary (CHO)-derived cells than did the native embryonic AChR in BC3H-1 cells, indicating that these two properties are determined by the host membrane and are not inherent properties of the AChR type. In addition, the differences in the macroscopic physical states of the lipids and membrane-associated solvent (water) dipolar relaxation between BC3H-1 and CHO-derived cells indicated by the spectroscopic properties of laurdan suggest that both lipid and associated water may influence the microscopic activity of individual AChR molecules embedded in the lipid bilayer. Finally, the different dependence of AChR channel conductance and mean open time as a function of GP observed between the different AChR subtypes in clonal cell lines suggests the importance of specific lipid-protein interactions in addition to bulk membrane properties.
Zanello
L P
LP
Instituto de Investigaciones Bioquímicas, Universidad Nacional del Sur, Bahia Blanca, Argentina.
Aztiria
E
E
Antollini
S
S
Barrantes
F J
FJ
D000818:Animals; D016466:CHO Cells; D002151:Calorimetry; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D002999:Clone Cells; D006224:Cricetinae; D018076:DNA, Complementary; D004553:Electric Conductivity; D004557:Electric Organ / Q000502:physiology; D004622:Embryo, Mammalian; D004625:Embryo, Nonmammalian; D015640:Ion Channel Gating; D008051:Lipid Bilayers; D051379:Mice; D018408:Patch-Clamp Techniques; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D014101:Torpedo; D014162:Transfection
D018076:DNA, Complementary; D008051:Lipid Bilayers; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins