1. Role of cytoplasmic residues in modulating voltage sensitivity of potassium channels
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 (Figure 1). The key functional elements identified are the inactivation gate, the voltage-sensing S4 transmembrane segment and the pore loop, which contains the selectivity filter. A major lacuna in our understanding of ion channel function is the manner in which movement of the voltage sensor (S4) is transduced into channel opening..

We have made a series of chimæric channels wherein this process is perturbed. In all the chimæras constructed, we have swapped cytoplasmic residues from either the N- or C-terminal tails, leaving the transmembrane portions intact. The most dramatic perturbation we have made is with the 4N/1T1 chimæra, where the operating voltage of the channel has been altered so that it is closed at resting membrane potentials but opens in a voltage-dependent manner on either depolarizing or hyperpolarizing, as opposed to the parental channels which open only on depolarization.

In another chimæric construct, the cooperativity of voltage-gated channel opening is perturbed. Most Kv channels undergo a gating transition over a 50 mV range, while in Kv 1.4 channels from a variety of species, this transition is spread over a range exceeding 100 mV. On swapping in the N-terminus from hKv1.1, the transition sharpens and is completed in less than 60 mV.

We will now attempt to identify the residues involved in these and other perturbations, in the hope that these residues form part of the machinery underlying the transduction.

Figure 1. Chimæric channels derived from two outwardly rectifying channels generate inward currents. Schematic representation of potassium channel constructs. a: hKv1.1; c: hKv1.4; e: Chimæra. The voltage sensing helix S4, the pore lining P-loop, the inactivating ball and the tetramerization domain T1 are highlighted. b, d and f: Currents elicited from T1 oocytes expressing these constructs. Oocytes were held at -80 mV and stepped to the -130 mV indicated potentials.

2. Towards a three dimensional structure of the potassium channel
Anurag Varshney and Baron Chanda

Detailed structural information is now available on three segments of the potassium channel: The pore itself; a region called the Tetramerization Domain or T1; and the Ball. However, it is not clear as to what spatial relationship these different segments bear to each other.

Based on the physiology of the 4N/1 series of chimæras and co-expression studies, we conclude that the T1 domain occludes the 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.

Figure 2. Elements of the potassium channel. Data from chimæric channels indicate that an intact T1 Domain prevents access of the “ball & chain” to the transmembrane surface, indicating that the “ball & chain” could be trapped within the basket formed by suspending the T1 tetramer below the transmembrane segment of the channel.

3. Voltage Dependent Anion Channel from rice
Ashwini Godbole

Programmed Cell Death is a critical feature in the development and maintenance of organisms from C elegans through plants to man. It appears that mitochondria may integrate various signals and initiate some death pathways by releasing proteins normally resident in the intermembrane space. We have overexpressed a Voltage Dependent Anion Channel (VDAC) from rice, osVDAC4, in mammalian cells. VDACs are â-barrel proteins that mediate the movement of ions and moderate-sized solutes across the outer membranes of mitochondria. Overexpression of osVDAC4 results in cell death with many characteristic features of apoptosis. Blocking the endogenous VDAC in these cells abrogates apoptotic death induced by stimuli such as staurosporine, which require mitochondrial involvement in their apoptotic progression. Mitochondrial permeability transition is not a requirement for death in these cases.

Figure 3a. Release of mitochondrial intermembrane proteins in apoptosis. Our data using reagents targeted to the Voltage Dependent Anion Channel (VDAC), and the Adenine Nucleotide Translocator (ANT) demonstrate that intermembrane proteins could be released from mitochondria without perturbing the inner membrane in response to some apoptotic stimuli. VDAC function is essential for such release suggesting that it could form part of the efflux conduit.
Figure 3b. Electrophysiology of osVDAC4. osVDAC4 was overexpressed in E coli and its ability to form ion channels studied using a planar bilayer setup (BLM). The trace represents VDAC channels closing on stepping from 0 mV to
-60 mV. 1M KCl was present on both sides of the membrane.

osVDAC4 purified from a bacterial expression system has been incorporated into bilayer lipid membranes and shown to form high conductance channels.

Collaborators: Apurva Sarin, NCBS and Usha VijayRaghavan, IISc.

4.Ion transport mechanisms underlying salt tolerance in plants
Veena Anil, K Sucharitha, K. Pannaga and Sam Kuruvilla

Plants utilize several strategies for surviving excessive salinity, some of which involve ion transport mechanisms. We are comparing salt-tolerant and sensitive cultivars of rice in an effort to identify such transporters.

We observed that at 2 weeks, the amount of Na+ that enters the shoot is dependent on calcium, suggesting a regulation of the ion specificity of Xylem Loading transporters. Of the Na+ that does enter the shoot, the salt-tolerant variety alone appears to sequester a significant amount in extracellular spaces. In older plants, an additional mechanism of sequestration in the root comes into play. We are currently following up on these observations and trying to elucidate the underlying mechanisms.

Collaborator: George Thomas, SPIC Science Foundation, Chennai.

Figure 4. Mechanisms that generate salt tolerance in rice. Top row, transverse shoot section of Pokkali, salttolerant coastal variety of rice. a, bright field image, b, scanning electron micrograph. Middle row, transverse shoot section of Jaya. c, bright
field image, d, scanning electron micrograph. Bottom row, e, percentage recovery of saline stressed plants plotted as a function of increasing NaCl-stress; f, total Na content in shoots plotted as a function of increasing medium Ca concentration (0-7 mM). Dark circle, Pokkali; Light circle, Jaya.