|RESEARCH REPORT 2001-2003
1. Using Chimæric Channels to Probe K + -channel Architecture
Anurag Varshney and S Kavitha
Voltage-gated potassium channels are among the most intensely studied proteins today. They are tetrameric proteins with each subunit consisting of six transmembrane segments and contributing a re-entrant “Pore-loop” to the pore.
We have constructed a series of chimæric channels with dramatically altered physiology. In particular, chimæras made by swapping N-terminal cytoplasmic residues between hKv1.1 and hKv1.4 mediate inward currents rather than the outward currents seen for the parent channels. The perturbation of the voltage sensing and transduction apparatus o f hKv1.1 by cytoplasmic residues from hKv1.4 has been exploited to determine the spatial arrangement of elements of the K + -channel. We conclude that the T1 domain occludes t he cytoplasmic loops, preventing interactions with large peptides. Our data strongly suggest that the “ball & chain” segments could be trapped within the basket formed by the T1 suspended below the transmembrane region during assembly (summary figure).
We are currently attempting to elucidate choreography within the protein: how changes in transmembrane potential lead to channel opening. Electrophysiology is being used to monitor movement of charges within the protein together with mutagenesis to perturb critical interactions.
2. Understanding Voltage transduction mechanism in Kv Channel
Sanjeev K Upadhyay
K+ channels undergo a voltage-dependent conductance change that plays a key role in modulating cellular excitability. While the open state is captured in the crystal structure, the losed state and the mechanism of this transition is still a subject of debate. We have built a model based on mutagenesis data and the gating currents measurement which is consistent with the idea that the open state is the default state, with field energy being used to keep the channel closed. Our model incorporates an “activated state” where the bulk of sensor movement is competed without channel opening. The model accounts for the well characterized electrophysiology of the V2 and ILT mutations, where sensor movement and channel opening occur over distinct voltage ranges. Moreover, it involves relatively small rearrangements in going from the activated to the open state, consistent with the rapid transitions observed in single channel records of Shaker type channels at zero millivolts.
3. Transporting a transporter: Regulation of hKv channels by a novel regulator, KCNRG
Voltage gated potassium channels are almost ubiquitously present in mammalian cells. They have been implicated in numerous cellular processes. Several auxiliary proteins have been shown to play regulatory roles in their function. The work focuses on one such protein, KCNRG (K+ Potassium CN Channel RG Regulator) which resides in the ER and modulates Kv surface levels.We propose KCNRG as a regulator of hKv's and potentially specific
to the Shaker family.
4. Voltage Dependent Anion Channel (VDAC) from Rice
We had previously demonstrated a critical role for the VDAC in the apoptotic release of cytochrome c from mitochondria. We have now overexpressed a rice VDAC in E coli, purified the protein, and reconstituted it into two model membrane systems. Reconstitution into liposomes allows an estimate of the pore diameter of the channel by monitoring the rate of transport of different-sized sugars through the pore. The Black Lipid Membrane (BLM) system is used to study the electrophysiological characteristics of the channel. Interestingly, we have observed a nearly non-conducting (20 pS) state of the channel at potentials comparable to those across the plasma membrane. Earlier reports on mammalian and Neurospora VDAC had characterized relatively high-conductance states (1 – 5 nS). We are now focusing on the manner in which channel conductance is regulated.
Collaborators: Apurva Sarin, NCBS and Usha Vijayraghavan, Indian Institute of Science, Bangalore.
5. Ion Transport Mechanisms Underlying Salt Tolerance In Plants
Veena Anil, K Sucharitha, K Pannaga and Sam Kuruvilla
Plants utilise several strategies for surviving excessive salinity, some of which may involve ion transport mechanisms. We have compared the manner in which salt-tolerant and – sensitive rice varieties handle NaCl stress. Survival is negatively correlated with Na in the apoplastic fluid surrounding cells. The primary response appears to be a Ca ++-dependent restriction of Na+ uptake into the shoots, followed by compartmentalisation within the shoot, probably within the large intracellular vacuoles. These mechanisms work well in the salt-tolerant variety, Pokkali, but are less efficiently deployed in the salt-sensitive variety, Jaya.
Collaborator: George Thomas, SPIC Science Foundation, Chennai.
6. Mechanisms Limiting Na+ levels in the cytosol of rice cells
We have used the ratiometric Na+ -sensitive dye, SBFI, to monitor Na+ levels in the cytosol of cells derived from Pokkali and Jaya and maintained in suspension culture. We find that Pokkali maintains low cytosolic Na+ even when medium Na+ is raised to 200 mM, whereas Jaya is unable to do so (Figure 3). The response is Ca++ –dependent. Our data implicate Na + /H+ - antiporters present in both the plasma membrane and the vacuolar membrane, pumping Na+ out of the cytosol. Efflux across the plasma membrane dominates this response.
Figure 1: Elements Of The Potassium Channel.
The voltage-gated K+ channel is a tetramer, each monomer having six transmembrane segments. In this figure, the front subunit has been removed and the first four segments are lumped together in the grey cylinders. A crystal structure of the pore-lining helices is shown along with the N-terminal T1 domain (bottom of the figure) and a “ball & chain” structure at its terminus.
Figure 2: Bilayer Lipid Membrane Studies on VDAC. A planar bilayer is painted across the pinhole separating the two aqueous compartments and currents across the membrane monitored at different set potentials. The panel at right shows currents recorded from a bilayer containing a single VDAC inserted. The potential was set to +70 mV. The different current levels are indicative of different conductance states of the channel. Note that the channel reaches a state that has a very low conductance.
Figure 3: Schematic of Fluid and Solute Transport in Rice. Fluid enters the rice plant through its roots, and is loaded i nto xylem elements in the root for onward transmission into the shoot. Fluid within the shoot is present inside cells in the cytoplasm and the vacuole, and also between cells in the apoplast.
Figure 4: Na+ Content of the Cytosol of Rice Cells in Suspension Culture. Rice Cells in suspension culture were loaded with SBFI, a ratiometric dye sensitive to Na +. The dye is restricted to the cytosol. On adding NaCl to 200 mM, the Na+ content of Jaya cells increases to more than 150 mM within 10–15 minutes (upper panel) with a corresponding increase in the fluorescence intensity of SBFI. The Na+ concentration in Pokkali cells rises to about 15 mM in the same time period (lower panel).