Historical Perspective of Pore-Forming Activity Studies of Voltage-Dependent Anion Channel (Eukaryotic or Mitochondrial Porin) Since Its Discovery in the 70th of the Last Century

Abstract

Eukaryotic porin, also known as Voltage-Dependent Anion Channel (VDAC), is the most frequent protein in the outer membrane of mitochondria that are responsible for cellular respiration. Mitochondria are most likely descendants of strictly aerobic Gram-negative bacteria from the α-proteobacterial lineage. In accordance with the presumed ancestor, mitochondria are surrounded by two membranes. The mitochondrial outer membrane contains besides the eukaryotic porins responsible for its major permeability properties a variety of other not fully identified channels. It encloses also the TOM apparatus together with the sorting mechanism SAM, responsible for the uptake and assembly of many mitochondrial proteins that are encoded in the nucleus and synthesized in the cytoplasm at free ribosomes. The recognition and the study of electrophysiological properties of eukaryotic porin or VDAC started in the late seventies of the last century by a study of Schein et al., who reconstituted the pore from crude extracts of Paramecium mitochondria into planar lipid bilayer membranes. Whereas the literature about structure and function of eukaryotic porins was comparatively rare during the first 10years after the first study, the number of publications started to explode with the first sequencing of human Porin 31HL and the recognition of the important function of eukaryotic porins in mitochondrial metabolism. Many genomes contain more than one gene coding for homologs of eukaryotic porins. More than 100 sequences of eukaryotic porins are known to date. Although the sequence identity between them is relatively low, the polypeptide length and in particular, the electrophysiological characteristics are highly preserved. This means that all eukaryotic porins studied to date are anion selective in the open state. They are voltage-dependent and switch into cation-selective substates at voltages in the physiological relevant range. A major breakthrough was also the elucidation of the 3D structure of the eukaryotic pore, which is formed by 19 β-strands similar to those of bacterial porin channels. The function of the presumed gate an α-helical stretch of 20 amino acids allowed further studies with respect to voltage dependence and function, but its exact role in channel gating is still not fully understood.

Keywords: VDAC; electrophysiology; eukaryotic pore; evolution; mitochondria; porin; single channel.

Figure 1

Relaxation of the membrane current in the presence of eukaryotic porin 1 from humans (hVDAC1, Porin 31HL; Benz et al., 1992). The membrane potential was first switched to +10mV and then to −10mV applied to the cis-side of the membrane containing about 50 hVDAC1 pores. Note that the membrane current did not decrease at these voltages. Then, higher positive and negative voltages were applied which resulted in a substantial exponential decrease of the membrane current. The membrane was formed of diphytanoyl phosphatidylcholine/n-decane. The aqueous phase contained 0.5M KCl (pH 7.2); T=20°C.

Figure 2

Ratio of the conductance, G, at a given voltage, V_m, divided by the conductance, G₀, at 10mV as a function of the voltage. The aqueous phase contained either 0.5M KCI, 0.5MK-MES, or 0.5M TRIS-HCI (pH in all cases 7.2). The cis-side contained about 10ng/ml hVDAC1 [Porin 31 HL (Benz et al., 1992)]. The sign of the voltage is given with respect to the cis-side, the side of the addition of Porin 31HL.

Figure 3

Semilogarithmic plot of the ratio, N₀/N_c, as a function of the transmembrane potential V_m. The data were taken from Figure 2. The slope of the straight lines is such that an e-fold change of N₀/N_c is produced by a change in V_m of 12.5mV (left side) and 11.9mV (right side), corresponding to gating charges, n=2.0 and 2.1, respectively. The midpoint potential of the N₀/N_c distribution (i.e., N₀=N_c) was at 27.4mV (left side) and 35mV (right side).

Figure 4

Stepwise increase of the membrane current (given in pA) after the addition of porin 31HL (hVDAC1) to a black lipid bilayer membrane given as a function of time. The aqueous phase contained 5ng/ml Porin 31HL and 1M KCl (Benz et al., 1992). The membrane was formed from diphytanoyl phosphatidylcholine/n-decane. The voltage applied was 10mV; T=20°C.

Figure 5

Histogram of conductance fluctuations observed with membranes of diphytanoyl phosphatidylcholine/n-decane in the presence of Porin 31HL (Benz et al., 1992). P(G) is the probability for the occurrence of a conductance step with a certain single-channel conductance (given in nS). The aqueous phase contained 1M KCl. The voltage applied was 10mV. The mean value of all upward directed steps was 4.3 nS for the right-side maximum and 2.4 nS for the left-side maximum (in total 288 single events); T=20°C.

Figure 6

Structure of the mitochondrial outer membrane pore (mVDAC1) and an OmpF monomer of E. coli. β-strands within the protein structures are shown in yellow and α-helical stretches in red. The 3D structures of the proteins are shown from the side in direction to the surface of the mitochondrion and the cell (upper structures) and from the surface of the mitochondrion and the bacterial cell (structures down). mVDAC1 (PDB code: 2JK4) is the 3D structure of mouse mitochondrial porin (Ujwal et al., 2008). OmpF (PDB code: 2OMF) represents the structure of the major outer membrane protein of E. coli (Cowan et al., 1992).